Pluripotent cell lines and methods of use thereof

ABSTRACT

Methods of generating cell lines with a sequence variation or copy number variation of a gene of interest, methods of use thereof, and cell lines with a sequence variation or copy number variation of a gene of interest are provided.

CROSS-REFERENCE

This application is a Continuation Application which claims the benefitof U.S. application Ser. No. 12/459,019, filed Jun. 24, 2009; whichclaims the benefit of U.S. Provisional Application No. 61/075,323, filedJun. 24, 2008, and U.S. Provisional Application No. 61/084,249, filedJul. 28, 2008, which applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The examination of the disease progression of complex diseases such asParkinson's disease is hindered by inaccessibility of neurons andlargely limited to use of postmortem samples. Primary cells frompostmortem brain samples have short life-spans in vitro and the diseaseitself reduces in number one cell type of interest—the midbrain dopamineneuron. To date, it has been virtually impossible to usedisease-affected neural tissues to screen for therapeutic agents usefulfor disease modification, predict disease progression and phenotype, andelucidate complex disease mechanisms. A model that replicates thefundamental features of the disease at the cellular level is needed.

Complex diseases such as Parkinson's disease, as well as many others,can involve genetic variations such as mutations and/or copy numbervariation (CNV) or can be idiopathic/sporadic. Induced pluripotent stemcells (iPSCs), developed from patients presenting with genetic andcomplex diseases, further differentiated into specific cell types, knownto be affected in the natural progression of disease, will providehertofore unavailable cellular tools for identification of diseasemechanisms, therapeutic agent screening, and disease diagnosis.

SUMMARY OF THE INVENTION

The invention described herein provides methods and compositions forgenerating induced pluripotent stem cells (iPSCs) specific to a diseaseof interest, wherein the iPSCs are further differentiated to adopt acell fate involved in the disease. The differentiated iPSCs can be usedfor screening, diagnostics, and the like. In a preferred embodiment,patient-specific iPSCs related to Parkinson's disease orParkinson's-like disease are generated. The patients can have a geneticform of the disease or a sporadic form of the disease. The Parkinson'sdisease and Parkinson's disease-related iPSCs are further differentiatedinto cell types, such as dopaminergic neurons, involved in theprogression disease. The differentiated iPSCs, relevant to Parkinson'sdisease and Parkinson's-like disease, are a valuable cellular model forelucidating basic disease mechanisms, screening for therapeutics, andfor use in diagnostic, prognostic, and theranostic applications.

In one aspect the invention described herein provides a method ofproducing dopaminergic cells from a patient having a diagnosis ofParkinson's disease or Parkinson's-like disease, the method comprisingobtaining fibroblasts from the patient, dedifferentiating thefibroblasts into pluripotent stem cells, and differentiating the stemcells towards a dopaminergic cell fate. In one aspect the inventiondescribed herein provides a method of producing dopaminergic cells froma patient having a diagnosis of Parkinson's disease or Parkinson's-likedisease, the method comprising obtaining fibroblasts from the patient,dedifferentiating the fibroblasts into pluripotent stem cells, anddifferentiating the stem cells towards a dopaminergic cell fate. In oneembodiment the fibroblasts are dermal fibroblasts. In another embodimentthe stem cells are differentiated towards a midbrain dopaminergic cellfate. In particular embodiments, the dedifferentiating of thefibroblasts induces pluripotency.

In some embodiments, the patient carries a genetic variation or mutationknown to be associated with Parkinson's disease or Parkinson's-likedisease. In other embodiments the genetic variation of interest is acopy number variation of a gene of interest. In specific embodiments,the cell line has three copies of the gene of interest. The geneticvariation or mutation can be a deletion, an insertion, a complexmulti-state variant, a deletion, a substitution, a transition, atransversion, or a duplication, of one or more nucleotides in the geneof interest. In exemplary embodiments, the gene of interest is selectedfrom PARK1 (SNCA or α-synuclein), PARK2 (Parkin), PARK5 (UCHL1), PARK6(PINK1), PARK7 (DJ-1), PARK8 (LRRK2), and PARK 11 (GIGFY2). In specificembodiments the gene of interest is PARK1 (SNCA or α-synuclein). Inother specific embodiments, the gene of interest is PARK8 (LRRK2). Inone embodiment the patient carries a homozygous mutation of LRRK2 andthe mutation comprises a G2019S mutation. In some embodiments, thepatient has Parkinson's disease. In a specific embodiment the patienthas an idiopathic form of Parkinson's disease.

In another aspect the invention described herein provides a method ofdetermining whether an agent is useful in the treatment of Parkinson'sdisease or Parkinson's-like disease, the method comprising contactingdopaminergic cells produced from induced pluripotent stem cells derivedfrom a patient with Parkinson's disease or Parkinson's-like disease,detecting the presence or absence of a response in the cells, anddetermining whether the agent is useful in the treatment based on thedetected response. In one embodiment, the detected response is a changein cell viability, cellular chemistry, cellular function, mitochondrialfunction, cell aggregation, cell morphology, cellular proteinaggregation, gene expression, cellular secretion, or cellular uptake. Ina related embodiment, the agent is selected from a small molecule, adrug, an antibody, a hybrid antibody, an antibody fragment, a siRNA, anantisense RNA, an aptamer, a protein, or a peptide. In a relatedembodiment, the agent corrects an α-synuclein dysfunction. In specificembodiments, the agent is selected from the group consisting ofapomorphine, pyrogallol, 1,4-naphthoquinone, cisplatin, isoproterenol,pyrogallin, cianidanol, sulfasalazine, quinalizarin, benserazide,hexachlorophene, pyrvinium pamoate, dobutamine, methyl-dopa, curcumin,berberine chloride, daidzein, merbromin, norepinephrine, dopaminehydrochloride, carbidopa, ethylnorepinephrine hydrochloride, tannicacid, elaidyphosphocholine, hydroquinone, chlorophyllide Cu complex Nasalt, methyldopa, isoproterenol hydrochloride, benserazidehydrochloride, dopamine, dobutamine hydrochloride, thyroid hormone,purpurin, sodium beta-nicotinamide adenine dinucleotide phosphate,lansoprazole, dyclonine hydrochloride, pramoxine hydrochloride,azobenzene, cefamandole sodium, cephaloridine, myricetin,6,2′,3′-trihydroxyflavone, 5,7,3′,4′,5′-pentahydroxyflavone,7,3′,4′,5′-tetrahydroxyflavone, (5,6,7,4′-tetrahydroxyflavone),baicalein, eriodictyol, 7,3′,4′-trihydroxyisoflavone, epigallocatechingallate, quercetin, gossypetin (3,5,7,8,3′,4′-hexahydroxyflavone),2′,3′-dihydroxyflavone, 3′,4′-dihydroxyflavone,5,6-dihydroxy-7-methoxyflavone, baicalein-7-methyl ether, 1-dopa, DOPAC,homogentisic acid, 6-hydroxydopamine, epinephrine, 3,4-dihydroxycinnamicacid, 2,3-dihydroxynaphthalene, 3,4-dihydroxybenzoic acid,3,4,5-trihydroxybenzoic acid, 1,2,3-trihydroxybenzoic acid, gallate(gallic acid), benzoquinone, catechol, rifampicin, rosmarinic acid,baicalin, tanshinones I and II, emodin, procyanidin B4, resveratrol,rutin, fisetin, luteolin, fustin, epicatechin gallate, catechin,alizarin, tannic acid, eriodyctol, carboplatin,purpurogallin-4-carboxylic acid, koparin,2,3,4-trihydroxy-4′-ethexybenzophenone, baeomycesic acid, hamtoxylin,iriginol hexaaceatate, 4-acetoxyphenol, theaflavin monogallate,theaflavin digallate, stictic acid, purpurogallin,2,5-dihydroxy-3,4-dimethoxy-4′-ethoxybenzophenone, promethazinehydrochloride, oxidopamine hydrochloride, pyrantel pamoate,elaidylphosphocholine, amphotericin B, gallic acid, fumarprotocetraricacid, theaflavin, haematoxylin pentaacetate, 4-methoxydalbergione,epigallocatechin-3-monogallate, rolitetracycline, 7,3′-dimethoxyflavone,liquiritigenin dimethyl ether, catechin pentaacetate, apigenin,3,4-dedesmethyl-5-deshydroxy-3′-ethoxyscleroin, derivatives and analogsthereof.

In one embodiment the induced pluripotent stem cells are derived fromhuman fibroblasts. In a related embodiment the induced pluripotent stemcells are produced without the use of a retrovirus or a lentivirus. In aspecific embodiment the induced pluripotent stem cells are produced witha method comprising the use of three factors. The three factors can beOCT4, SOX2, and KLF4. In another embodiment, the induced pluripotentstem cells are further differentiated to adopt a midbrain dopaminergiccell fate. In some cases, the induced pluripotent stem cells aredifferentiated to adopt the cell fate in about 20 days.

In another embodiment, the patient carries a genetic variation ormutation known to be associated with Parkinson's disease orParkinson's-like disease. In a related embodiment the genetic variationof interest is a copy number variation of a gene of interest. Inspecific embodiments, the cell line has three copies of the gene ofinterest. The genetic variation or mutation can be a deletion, aninsertion, a complex multi-state variant, a deletion, a substitution, atransition, a transversion, or a duplication, of one or more nucleotidesin the gene of interest. In a specific embodiment the gene of interestis selected from PARK1 (SNCA or α-synuclein), PARK2 (Parkin), Park5(UCHL1), PARK6 (PINK1), PARK7 (DJ-1), PARK8 (LRRK2), and PARK 11(GIGFY2). In related embodiments the gene of interest is PARK1 (SNCA orα-synuclein). In other related embodiments, the gene of interest isPARK8 (LRRK2). In a specific related embodiment, the patient carries ahomozygous mutation of LRRK2. The LRRK2 mutation can comprise a G2019Smutation. In some embodiments the patient has Parkinson's disease. Inother embodiments, the patient has an idiopathic form of Parkinson'sdisease.

In yet another aspect, the invention provides for an induced pluripotentcell line from a patient with Parkinson's disease or Parkinson's-likedisease, produced by obtaining fibroblasts from a patient withParkinson's disease or Parkinson's-like disease carrying a geneticmutation which causes the disease, dedifferentiating the fibroblasts,whereby producing induced pluripotent stem cells, and differentiatingthe stem cells towards a dopaminergic cell fate, whereby producing aninduced pluripotent cell line from a patient with Parkinson's disease orParkinson's-like disease. In one embodiment, the fibroblasts are dermalfibroblasts. In another embodiment the pluripotent stem cells aredifferentiated towards a midbrain dopaminergic cell fate. In a relatedembodiment the patient carries a genetic variation or mutation known tobe associated with Parkinson's disease or Parkinson's-like disease. In aspecific embodiment, the genetic variation of interest is a copy numbervariation of a gene of interest. In a related specific embodiment, thecell line has three copies of the gene of interest. In another relatedembodiment the genetic variation or mutation is a deletion, aninsertion, a complex multi-state variant, a deletion, a substitution, atransition, a transversion, or a duplication, of one or more nucleotidesin the gene of interest. In one embodiment the gene of interest isselected from PARK1 (SNCA or α-synuclein), PARK2 (Parkin), PARK5(UCHL1), PARK6 (PINK1), PARK7 (DJ-1), PARK8 (LRRK2), and PARK 11(GIGFY2). In another embodiment the gene of interest is PARK1 (SNCA orα-synuclein). In a specific embodiment the gene of interest is PARK8(LRRK2). In a specific related embodiment, the patient carries ahomozygous mutation of LRRK2, with a G2019S mutation. In one embodimentthe patient has Parkinson's disease. In another embodiment the patienthas an idiopathic form of Parkinson's disease. In a related embodiment,the induced pluripotent cell line is produced without the use of aretrovirus or a lentivirus, or produced with a method comprising the useof three factors. The three factors can be OCT4, SOX2, and KLF4. In arelated embodiment, the induced pluripotent cell line is differentiatedto adopt the cell fate in about 20 days.

In a related aspect the invention provides for a human inducedpluripotent cell line from a patient with Parkinson's disease whereinthe cell line comprises a genetic variation related to Parkinson'sdisease. In one embodiment, the variation is triplication of the SNCAgene. In another embodiment, the variation is a homozygous mutation ofthe LRRK2 gene. In a specific embodiment, the induced pluripotent cellline is produced from fibroblasts of a patient with Parkinson's disease.In another embodiment the induced pluripotent cell line is producedwithout the use of a retrovirus or a lentivirus or with a methodcomprising the use of three factors. The three factors can be OCT4,SOX2, and KLF4. In a specific embodiment, the induced pluripotent cellline is further differentiated to adopt a midbrain dopaminergic cellfate. In a related embodiment, the induced pluripotent cell line isdifferentiated to adopt the cell fate in about 20 days.

In yet another related aspect, the invention provides a human inducedpluripotent cell line from a patient with Parkinson's disease, whereinthe cell line comprises either a genetic variation comprising avariation in the SNCA gene or the LRRK2 gene.

Methods of generating cell lines with genetic variations of a gene ofinterest, methods of use thereof, and cell lines with sequence and/orcopy number variations of a gene of interest are also provided herein.

In some aspects, the methods include providing cells with sequencevariations or multiple copies of the gene of interest, and inducingpluripotency, multipotency or totipotency in the cells to make a cellline with the variation of the gene of interest. The methods may alsoinclude identifying a subject with a variation of the gene of interestand obtaining one or more cells from the subject. The subject-derivedcells may be fibroblast cells, tumor cells, bone marrow cells, stomachcells, blood cells (such as white blood cells, blood progenitor cells),liver cells, etc. or any convenient or relevant source of cells to beobtained from the subject. The methods of generating cell lines with acopy number variation of a gene of interest may also include inducingdifferentiation of the cell line. The cell lines may be differentiatedinto any cell type of interest including endodermal, ectodermal,mesodermal, for example neural, such as neuronal cell lines, epithelialcell lines, cardiac cell lines, etc.

In some aspects, the cell lines and methods of their use include celllines with a genetic copy number variation that is at least oneduplication, for example, two or three multiple copies of a gene. Inother aspects, the copy number variation is one or more deletion,insertion, or complex multi-state variant of the gene of interest.

In some aspects, the cell lines and methods of their use include celllines with a genetic variation that is a mutation of the gene ofinterest. For example, the mutation may be a deletion, substitution,transition, transversion, or duplication of one or more nucleotides inthe gene of interest. In some embodiments, the mutation is a pointmutation.

In some aspects, the cell lines and methods of their use include celllines with a genetic variation that is associated with a disorder, suchas a genetic disease.

Methods of screening cell lines with variations of a gene of interestfor an agent to treat a disorder or disease are also provided herein.The methods include contacting the agent with a cell line or its progenymade by the methods described herein, observing a change or lack ofchange in the cells, and correlating a change or lack of change with theability of the agent to treat the disease. Such changes may be observed,for example, by staining for one or more markers. The methods mayfurther comprise comparing the cell line or its progeny with a cell lineor its progeny without the variation in the gene of interest, that is, anormal cell line, or a cell line correlated to the same disorder ofinterest, but without the variation in the gene of interest present inthe first cell line.

Methods of studying the mechanism of a disease of interest are alsoprovided herein. The methods include identifying a molecular determinantof the disorder or disease by contacting a cell line or its progeny madeby the methods described herein with an agent or condition which affectsa cellular pathway of interest and observing a change or lack of changein the cells.

Methods of treating or preventing disorders are also provided herein.The methods include administering to a subject an agent identified bythe screening methods described herein an effective amount to treat orprevent the disorder. Disorders include diseases and susceptibilities,for example, Parkinson's disease, Alzheimer's disease, dementia, anautism spectrum disorder, susceptibility to viral infection, diffuseLewy body disease or any other Lewy body disorder or synucleinopathy,corticobasal degeneration, encephalitis lethargica, multiple systematrophy, pantothenate kinase-associated neurodegeneration(Hallervorden-Spatz syndrome), progressive supranuclear palsy, vascularParkinsonism, Wilson disease, hereditary pancreatitis,glomerulonephritis, human systemic lupus erythematosus, paraneoplasticsyndrome, frontotemporal dementia with Parkinsonism chromosome 17,Huntington's disease, spinocerebellar ataxias, amytropic lateralsclerosis, Creutzfeld Jakob disease, and other conditions resulting fromgenetic variations.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of derivation of primary humanfibroblasts from human subjects.

FIG. 2 illustrates (A) Human fibroblast cells (HUF1 cell line); middlepanel shows expression of GFP from the ubiquitin promoter; (B)expression of OCT4 post-infection with a lentivirus:OCT4 cDNA; (C)generation of colonies; (a) no factors, (b) Yamanaka factors withgranular colony, (c) Thomson factors with hESC-like colony.

FIG. 3 illustrates hESCs subjected to 49 days of patterning anddifferentiation to generate cultures containing dopaminergic neurons.(A) Differentiation involved a multi-step procedure where cells arefirst neutralized in embryoid body microaggregates and then plated undermidbrain induction conditions that include FGF-8 and sonic hedgehog.Further differentiation leads to cultures containing aggregates of NeuNand doublecortin (Dcx) positive neurons (B,C), many of which aretyrosine hydroxlase positive (TH). Nuclei are stained with DAPI. (D) and(E) are magnifications of boxed area in the lower left of panel (C).

FIG. 4 illustrates the expression of GFP in primary human fibroblasts(HUF1 cell line) following transduction with FUGW UbC-GFP lentivirus.

FIG. 5 illustrates the morphologies of the reprogrammed/dedifferentiatedcell lines.

FIG. 6 illustrates the morphologies of the reprogrammed/dedifferentiatedcell lines.

FIG. 7 illustrates the morphologies of the reprogrammed/dedifferentiatedcell lines.

FIG. 8 illustrates gene dosage of Exon 3 of the SNCA gene and confirmsSNCA triplication in the HUF4 iPSC clone 17 line, as assessed by qPCR.

FIG. 9 illustrates the untransduced fibroblasts used for the generationof the HUF4 and HUF5 lines.

FIG. 10 illustrates HUF4 (clone 17) phase contrast images of morphologyon MEF feeder layer at 10×, 5× and 20× (Panels 1-3), alkalinephosphatase staining (Panel 4), nuclear staining with DAPI andimmunocytochemistry of pluripotency factors; SSEA, Tra1-60, TRA1-81,NANOG, SSEA4, and SSEA1.

FIG. 11 illustrates HUF5 (clone 2) phase contrast images of morphologyon MEF feeder layer at 10×, 5× and 20× (Panels 1-3), alkalinephosphatase staining (Panel 4), nuclear staining with DAPI andimmunocytochemistry of pluripotency factors; SSEA, Tra1-60, TRA1-81,NANOG, SSEA4, and SSEA1.

FIG. 12 illustrates in vitro differentiation of HUF4 (clone 17) (leftpanel) and HUF5 (clone 2) (right panel) to embryoid bodies, functionalcardiac myocyte differentiation and the three germ layers; endoderm(α-Fetalprotien), mesoderm (α-SMA and Desmin) and ectoderm(β-III-Tubulin, DCX and TH).

FIG. 13 illustrates a karyotype analysis of HUF4 (clone 17) and HUF5(clone 2) derived iPSC lines. 20 replicates showed no translocations,triplications, or deletions.

FIG. 14 illustrates the methylation status of Nanog and Oct4 promotersas determined by bisulfite sequencing in untransduced fibroblasts (HUF4clone 17 p8 and HUF5 clone 2 p6) and pluripotent iPSC lines (HUF4-iPSand HUF5-iPS)/

FIG. 15 illustrates the relative telomerase activity in: untransducedfibroblast lines (HUF4 and HUF5); pluripotent iPSC lines HUF4 (clonesc17, c46) and HUF5 (clones c2 and c3); pluripotent hESC line HSF8;differentiated iPSC lines HUF4 (c17) and HUF5 (c2); negative buffer onlycontrol; and TSR8 positive control.

FIG. 16 illustrates HUF4 (clone 17) teratoma sectioning pathology.Formation of the endoderm is exemplified by the formation of theepithelial mucin and gut-like epithelium; formation of the mesoderm isexemplified by formation of the smooth muscle and cartilage; andformation of the ectoderm is exemplified by formation of neural tissue.

FIGS. 17-21 illustrate expression of endogenous and exogenoustranscription factors following reprogramming in HUF4 and HUF 5 lines(parental, iPSCs, differentiated, different clones) as well as controlH9 and Hela cells. Endogenous and exogenous expression of Oct4, Sox2,cMyc, and Klf4 expression are examined.

FIG. 22 illustrates neural induction at day 28 (phase contrast images)and day 50 (neural immunocytochemistry images for TH and NESTIN; H9 (Aand D), HUF4 clone 17 (B and E) and HUF5 clone 2 (C and F). TH positivecells show the presence of dopaminergic neurons in culture, while lackof colocalization between TH and NESTIN indicate olfactory bulb neuronabsence and midbrain dopaminergic neuron presence.

FIG. 23 illustrates HUF4 clone 17 neural induction time course phasecontrast images 5× isolocation (except where noted): Days 3, 6, 9, 9(left-inferior), 12 (left-inferior 10×), 14 (left-inferior). Formationof neural rosettes are visible begging on day 9 and continue to expandand proliferate through day 14 and beyond.

FIG. 24 illustrates HUF5 clone 2 neural induction time course phasecontrast images 5× isolocation (except where noted): Days 3, 6, 9, 9(left-inferior), 12 (left-inferior 10×), 14 (left-inferior).

FIG. 25 illustrates somatic gene expression in H9 ESCs, anddifferentiated HUF4 and HUF5 iPSCs.

FIG. 26 illustrates α-synuclein staining of HUF4 cells.

FIG. 27 illustrates the G2019S mutation in the HUF6, but not HUF4 lines.

FIG. 28 illustrates the generation of the HUF6 iPSC line with 3 factors:Oct4, Sox2, and Klf4. The figure illustrates staining with pluripotencymarkers.

FIG. 29 illustrates the spectral karyotype analysis of the HUF6 iPSCline.

FIG. 30 shows growth and expansion of HUF6 iPS cells on matrigel andfeeders.

FIG. 31 illustrates differentiation of the HUF6 iPSCs to dopaminergicneurons using a 20-day protocol. The figure presents images of the cellsduring the time course of differentiation to dopaminergic neurons.

FIG. 32 illustrates the differentiation of HUF6-iPSCs into dopaminergicneurons after 20 days.

FIG. 33 illustrates that specific agents can be used to block or reversepathological phenotypes such as α-synuclein aggregation. The figureshows in vitro and ex vivo methods for rapid screening ofanti-aggregation compounds: (A) Inhibition of fibrillation ofα-synuclein upon incubation with a specific inhibitor, detected byThioflavin T fluorescein and (B-C) confirmed by electron microscopy ofα-synuclein fibrils. (D-E). Diminished Thioflavin S deposits (whichlabel aggregate protein) detected in paraquat-treated mouse brain withCatechol (250 uM), indicative of treatment-induced dissolution ofaggregate structure.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION Creation of Induced PluripotentStem Cell Lines Specific to Diseases and Disorders

Overview:

Methods of generating cell lines with genetic variations of a gene ofinterest, methods of use thereof including differentiation of the cellsto a lineage of interest, the use of cell lines in identifying agents totreat a disorder, use of cell lines in studying mechanism of disease,and cell lines with sequence and/or copy number variations of a gene ofinterest are provided herein. In preferred embodiments, these methodsare used to generate Parkinson's disease-specific cell lines, for use inelucidating Parkinson's disease mechanisms, diagnosis of Parkinson'sdisease, and screening for agents useful in the treatment or preventionof Parkinson's disease.

Starting with cells, such as human cells, induced pluripotent stem (iPS)cells are generated, for example, using combinations of transcriptionfactors. In some embodiments, human cells obtained from a subject, forexample, for the purpose of inducing dedifferentiation may be chosenfrom any human cell type, including fibroblast cells, tumor cells, bonemarrow cells, stomach cells, liver cells, epithelial cells, nasalepithelial cells, mucosal epithelial cells, follicular cells, connectivetissue cells, muscle cells, bone cells, cartilage cells,gastrointestinal cells, splenic cells, kidney cells, lung cells,testicular cells, nervous tissue cells, and lymphocytic cellstransformed with Epstein-Barr virus. In some embodiments, the human celltype is a fibroblast, which may be conveniently obtained from a subjectby a punch biopsy. In certain embodiments, the cells are obtained fromsubjects known or suspected to have a copy number variation (CNV) ormutation of the gene of interest. In other embodiments, the cells arefrom a patient presenting with idiopathic/sporadic form of the disease.In yet other embodiments, the cells are from a non-human subject. Thecells are then differentiated to adopt a specific cell fate, such asneuronal cells, for example dopaminergic, cholinergic, serotonergic,GABAergic, or glutamatergic neuronal cell fates.

Somatic cells, with a combination of three, four, five, six, or morefactors can be dedifferentiated/reprogrammed to a state apparentlyindistinguishable from embryonic stem cells (ESCs); these reprogrammedcells are termed “induced pluripotent stem cells” (iPSCs, iPCs, iPSCs)and can be produced from a variety of tissues.

The factors appear to reverse epigenetic landmarks thereby introducingpluripotency in the cells. iPSCs possess the ability to differentiateinto the cell types of all three germ layers; in the mouse, contributionto the germ line has been observed. Therefore, patient-specific celllines can be established for various applications, including the studyof disease mechanisms and screening for potential therapeutics.

Using the methods described herein, human iPSCs may be generated frompatients with specific diseases, for example Parkinson's disease, forexample due to a triplication of the SNCA gene or due to a mutation inthe LRRK2 gene and used for further applications such as screening ordisease diagnostics.

Dedifferentiation/Reprogramming of Cells to Induced Pluripotency:

Differentiation of a cell is the process by which cells becomestructurally and functionally specialized, for example, during embryonicdevelopment or in vitro. By dedifferentiation or reprogramming, cellsare restored to an unspecialized state. Dedifferentiation allows forrespecialization into other cell types distinct from that of the cellwhich has undergone the dedifferentiation.

Once obtained, cells may be dedifferentiated by exposure totranscription factors such as, for example OCT4, SOX2, KL4, andoptionally cMyc, NANOG, and/or LIN28. The induction of dedifferentiationincludes exposing cells to characterized factors which are known toproduce a specific lineage outcome in cells exposed to the factors, soas to target the dedifferentiation to that of a specific desired lineageand/or cell type of interest. The exposure to transcription factors maybe accomplished, e.g., by viral or non-viral method of application ortransduction. The outcome of the dedifferentiation is a cell line, i.e.,a cell or cells that have the capacity to be propagated indefinitely orfor long periods of time, including months to years in continuousculture.

Dedifferentiation is the induction of pluripotency, multipotency, ortotipotency, whereby a cell is restored to a more undifferentiatedcondition such as pluripotency (having the potential to differentiateinto any of the three germ layers (endoderm, ectoderm, mesoderm));multipotency (having the potential to differentiate into some, but notall, tissue types); or totipotency (having the potential to rise to allthe cell types that make up an organism plus all of the cell types thatmake up the extraembryonic tissues, such as the placenta). Using theestablished dedifferentiation protocols of Takahashi and others, one ofskill in the art may produce a pluripotent cell and differentiatetherefrom any cell derived from the three primary germ layers. One ofskill in the art will appreciate, however, that, depending upon thecells obtained from the subject which are to be dedifferentiated, thedesired cell line, and the desired lineage following differentiation ofthe cell line, varying degrees of dedifferentiation may be sufficient,where the varying degrees of dedifferentiation may optionally beeffected by, for example, exposure of the cells to differentcombinations of transcription factors, the use of a combination oftranscription factors in different proportions, exposure thereto fordifferent periods of time, etc., such that full dedifferentiation is notperformed where it is not required to produce the desired cell line.However, the protocols of Takahashi and others as discussed may beutilized to give rise to endodermal, ectodermal or mesodermal celllines, or their progeny.

This invention can also be practiced using stem cells of various types,which may include the following non-limiting examples.

U.S. Pat. No. 5,851,832 reports multipotent neural stem cells obtainedfrom brain tissue. U.S. Pat. No. 5,766,948 reports producing neuroblastsfrom newborn cerebral hemispheres. U.S. Pat. Nos. 5,654,183 and5,849,553 report the use of mammalian neural crest stem cells. U.S. Pat.No. 6,040,180 reports in vitro generation of differentiated neurons fromcultures of mammalian multipotential CNS stem cells. WO 98/50526 and WO99/01159 report generation and isolation of neuroepithelial stem cells,oligodendrocyte-astrocyte precursors, and lineage-restricted neuronalprecursors. U.S. Pat. No. 5,968,829 reports neural stem cells obtainedfrom embryonic forebrain and cultured with a medium comprising glucose,transferrin, insulin, selenium, progesterone, and several other growthfactors.

Except where otherwise required, the invention can be practiced usingstem cells or induced pluripotent stem cells of any vertebrate species.Included are stem cells from humans; as well as non-human primates,domestic animals, livestock, and other non-human mammals.

Among the stem cells suitable for use in this invention are mammalianpluripotent and multipotent stem cells derived from tissue formed aftergestation, such as a blastocyst, or fetal or embryonic tissue taken anytime during gestation. Non-limiting examples are primary cultures orestablished lines of embryonic stem cells or embryonic germ cells.

Establishment of Embryonic Stem Cell Lines:

Embryonic stem cells can be isolated from blastocysts of members of theprimate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844,1995). Human embryonic stem (hES) cells can be prepared from humanblastocyst cells using the techniques described by Thomson et al. (U.S.Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133ff., 1998) and Reubinoff et al, Nature Biotech. 18:399, 2000.

Briefly, human blastocysts are obtained from human in vivopreimplantation embryos. Alternatively, in vitro fertilized (IVF)embryos can be used, or one-cell human embryos can be expanded to theblastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos arecultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner etal., Fertil. Steril. 69:84, 1998). The zona pellucida is removed fromdeveloped blastocysts by brief exposure to pronase (Sigma). The innercell masses are isolated by immunosurgery, in which blastocysts areexposed to a 1:50 dilution of rabbit anti-human spleen cell antiserumfor 30 min, then washed for 5 min three times in DMEM, and exposed to a1:5 dilution of Guinea pig complement (Gibco) for 3 min (Solter et al.,Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two further washes inDMEM, lysed trophectoderm cells are removed from the intact inner cellmass (ICM) by gentle pipetting, and the ICM plated on mEF feeder layers.

After 9 to 15 days, inner cell mass-derived outgrowths are dissociatedinto clumps, either by exposure to calcium and magnesium-freephosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispaseor trypsin, or by mechanical dissociation with a micropipette; and thenreplated on mEF in fresh medium. Growing colonies havingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and replated. ES-like morphologyis characterized as compact colonies with apparently high nucleus tocytoplasm ratio and prominent nucleoli. Resulting ES cells are thenroutinely split every 1-2 weeks by brief trypsinization, exposure toDulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase(.about.200 U/mL; Gibco) or by selection of individual colonies bymicropipette. Clump sizes of about 50 to 100 cells are optimal.

Genetic Variations:

The methods described herein include dedifferentiation of cells in orderto produce a cell line with a genetic variation. Genetic variationsinclude differences in DNA sequence (within coding and/or non-codingregions); differences in gene expression and heritable variances thatexist among members of a species; polymorphisms, including, for example,nucleotide polymorphisms, restriction site polymorphisms, transitions,transversions, deletions, insertions, and duplications of one or morenucleotides; deletions, insertions, transpositions, and duplications ofone or more genes; and gene copy number variations. Other types ofgenetic variations are known in the art.

In some embodiments, the genetic variation of interest is a copy numbervariation (CNV), also known as a copy number polymorphism (CNP). CNVencompasses the variation of the number of copies of a gene, or ofsequences of DNA, in the genome of an individual. The CNV of the gene ofinterest may be of any type. Nonlimiting examples include duplication,triplication, deletion, insertion, complex multi-state variant of thegene of interest, and combinations thereof. Copy number variation hasbeen linked to disease susceptibility and disease resistance. The geneof interest may be any gene known or suspected to have a CNV or othergenetic variation such as a mutation. Genes known to have a CNVs ormutations include α-synuclein (PARK1, SNCA), Parkin (PARK2), UCHL1(PARK5), PINK1 (PARK6), DJ-1 (PARK7), LRRK2 (PARK5), GIGFY2 (PARK 11),SLC4A10, FHIT, FHIT, FLJ16237, A2BP1, and CCL3L1. One of skill in theart can readily determine which genes have a CNV using known techniques,or by reference to published sources.

For example, the Wellcome Trust Sanger Institute has developed DECIPHER(Database of Chromosomal Imbalance and Phenotype in Humans Using EnsemblResources), a database of CNVs associated with clinical conditions,available online at https://decipher.sanger.ac.uk/. See also Lupski, J,Genome structural variation and sporadic disease traits, Nat. Genet. 38,974-76 (2006). Foundations related to research on a disease conditionfrequently make available databases of genes associated with thedisease. In the case of Parkinson's disease, the Michael J. FoxFoundation provides access to a comprehensive and regularly updatedcollection of genetic association studies performed on Parkinson'sdisease (PD) phenotypes, accessible by entering in a browser the URLhttp://www(dot)pdgene(dot)org/.

In further embodiments, the genetic variation of interest may be avariation in the nucleotide sequence of a gene, i.e., a mutation. Thevariation in the nucleotide sequence of the gene of interest includesany alteration in a polynucleotide and may be of any type. Nonlimitingexamples include a deletion, substitution, transition, transversion, orduplication of one or more nucleotides in the gene of interest, andcombinations thereof. One of skill in the art can readily determinewhich genes have a mutation of interest using known techniques, or byreference to published sources. For example, databases related todiseases associated with genetic mutations accessible by entering a URLinto a browser include:medicalgenetics(dot)med(dot)ualberta(dot)ca/wilson/index(dot)php,molgen(dot)ua(dot)ac(dot)be/ADMutations/,bioinf(dot)uta(dot)fi/KinMutBase/,www(dot)humgen(dot)rwth-aachen(dot)de/index(dot)asp?subform=database(dot)html&nav=database_nav(dot)html,cooke(dot)gsf(dot)de/asthmaGen, life2(dot)tau(dot)ac(dot)il/GeneDis/,fmf(dot)igh(dot)cnrs(dot)fr/infevers/, imgt(dot)cines(dot)fr,www(dot)tmgh(dot)metro(dot)tokyo(dot)jp/jg-snp/english/E_top(dot)html,mutview(dot)dmb(dot)med(dot)keio(dot)ac(dot)jp/MutationView/jsp/mutview/index(dot)jsp,www(dot)med(dot)mun(dot)ca/mmrvariant/,www(dot)thepi(dot)org/altruesite/files/parkinson/Mutations/new_page_(—)1(dot)html,www(dot)pdgene(dot)org/,www(dot)retina-international(dot)org/sci-news/mutation(dot)htm,www(dot)ucl(dot)ac(dot)uk/ncl/mutation(dot)shtml,pkdb(dot)mayo(dot)edu/,www(dot)pathology(dot)washington(dot)edu/research/werner/database/ andwww(dot)genet(dot)sickkids(dot)on(dot)ca/cftr/Home(dot)html.

In some embodiments, the present methods include identifying a subjectwith a genetic variation of interest, and obtaining one or more cellsfrom the subject to obtain one or more cells with the genetic variationof interest. Suitable subjects include, for example, any species fromwhich at least one cell can be utilized for dedifferentiation, such ashumans, non-human primates, and non-primate mammals such as (but notlimited to) rodents. Genetic variations of interest may include, forexample, genetic variations associated with diseases, disorders, orantigens of interest.

In some embodiments, the genetic variation of interest is associatedwith a disorder, e.g., a disease. The genetic variation may be avariation in a gene or gene product, cellular pathway, etc., which iscausative, symptomatic, or diagnostic of any disease, disorder, illnessand the like. Disease-associated genes include, but are not limited to,a gene, a genomic region about 10 kb upstream and 10 kb downstream ofsuch gene, regulatory regions that modulate the expression of the gene,and all associated gene products (e.g., isoforms, splicing variants,and/or modifications, derivatives, etc.). The sequence of adisease-associated gene may contain one or more disease-related sequencepolymorphisms. For example, the sequence of a Parkinson'sdisease-related gene in an individual may contain one or more reference(i.e. “normal”) or alternate alleles, or may contain a combination ofreference and alternate alleles, or may contain alleles in linkagedisequilibrium with one or more polymorphic regions. The geneticvariation of interest may include a CNV or a variation in the nucleotidesequence of a gene, i.e., a mutation. One of skill in the art canreadily identify genetic variations associated with a disorder usingknown techniques, or by reference to published sources.

In some embodiments, the subject with the genetic variation of interesthas been diagnosed with a disorder or a predisposition to a disorder. Insome embodiments, the subject has been diagnosed with a genetic disorderor a predisposition to a genetic disorder. In certain embodiments, thesubject has been diagnosed with a neurodegenerative disorder. Disordersinclude, but are not limited to, Parkinson's disease (PD), PD-relateddiseases, toxicity-induced Parkinsonism, Alzheimer's disease, dementia,an autism spectrum disorder, susceptibility to viral infection such asHIV, and CHARGE Syndrome. Autism spectrum disorders include Aspergersyndrome, autism, pervasive developmental delay (PDD) not otherwisespecified, and Rett disorder. A PD-related disease refers to one or morediseases, conditions or symptoms or susceptibilities to diseases,conditions or symptoms, that involve, directly or indirectly,neurodegeneration including but not limited to the following:Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Alpers'disease, Batten disease, Cockayne syndrome, corticobasal ganglionicdegeneration, Huntington's disease, Lewy body disease, Pick's disease,motor neuron disease, multiple system atrophy, olivopontocerebellaratrophy, Parkinson's disease, postpoliomyelitis syndrome, priondiseases, progressive supranuclear palsy, Rett syndrome, Shy-Dragersyndrome and tuberous sclerosis. In certain aspects, a PD-relateddisease is a neurodegenerative disease the affects neurons in the brain.A PD-related disease may be e.g. a condition that is a risk factor fordeveloping PD, or may be a condition for which PD is a risk factor, orboth.

Other known disorders which are related to CNV wherein the presentmethods may find use include but are not limited to: 12q14 microdeletionsyndrome, 15q13.3 microdeletion syndrome, 15q24 recurrent microdeletionsyndrome, 16p11.2-p12.2 microdeletion syndrome, 17q21.3 microdeletionsyndrome, 1p36 microdeletion syndrome, 1q21.1 recurrent microdeletion,1q21.1 recurrent microduplication, 1q21.1 susceptibility locus forThrombocytopenia-Absent Radius (TAR) syndrome, 22q11 deletion syndrome(Velocardiofacial/DiGeorge syndrome), 22q11 duplication syndrome,22q11.2 distal deletion syndrome, 22q13 deletion syndrome(Phelan-Mcdermid syndrome), 2p15-16.1 microdeletion syndrome, 2q33.1deletion syndrome, 2q37 monosomy, 3q29 microdeletion syndrome, 3q29microduplication syndrome, 6p deletion syndrome, 7q11.23 duplicationsyndrome, 8p23.1 deletion syndrome, 9q subtelomeric deletion syndrome,adult-onset autosomal dominant leukodystrophy (ADLD), Angelman syndrome(Type 1), Angelman syndrome (Type 2), ATR-16 syndrome, AZFa, AZFb,AZFb+AZFc, AZFc, Cat-Eye Syndrome (Type I), Charcot-Marie-Tooth syndrometype 1A (CMT1A), Cri du Chat Syndrome (5p deletion), early-onsetAlzheimer disease with cerebral amyloid angiopathy, familial adenomatouspolyposis, hereditary liability to pressure palsies (HNPP), Leri-Weilldyschondrostosis (LWD)-SHOX deletion, Miller-Dieker syndrome (MDS),NF1-microdeletion syndrome, Pelizaeus-Merzbacher disease, Potocki-Lupskisyndrome (17p11.2 duplication syndrome), Potocki-Shaffer syndrome,Prader-Willi syndrome (Type 1), Prader-Willi syndrome (Type 2), RCAD(renal cysts and diabetes), Rubinstein-Taybi syndrome, Smith-Magenissyndrome, Sotos syndrome, split hand/foot malformation 1 (SHFM1),steroid sulphatase deficiency (STS), WAGR 11p13 deletion syndrome,Williams-Beuren syndrome (WBS), Wolf-Hirschhorn syndrome, and Xq28(MECP2) duplication.

In some embodiments, the subject with the genetic variation of interesthas been diagnosed with a disorder or predisposition to a disorderassociated with protein aggregation. Protein aggregation includesnonspecific coalescence of misfolded proteins, driven by interactionsbetween solvent-exposed hydrophobic surfaces that are normally buriedwithin a protein's interior. Normal interactions between misfoldedproteins with normal cellular constituents have been proposed tounderlie the toxicity associated with protein aggregates in manyneurodegenerative disorders. Such disorders include, for example,Alzheimer's disease, Parkinson's disease, dementia, autism spectrumdisorders, susceptibility to viral infection, diffuse Lewy body disease,other Lewy body disorders, synucleinopathy, corticobasal degeneration,encephalitis lethargica, multiple system atrophy, pantothenatekinase-associated neurodegeneration (Hallervorden-Spatz syndrome),progressive supranuclear palsy, vascular Parkinsonism, Wilson disease,hereditary pancreatitis, glomerulonephritis, human systemic lupuserythematosus, paraneoplastic syndrome, frontotemporal dementia withParkinsonism chromosome 17, Huntington's disease, spinocerebellarataxias, amytropic lateral sclerosis, and Creutzfeldt-Jakob disease.

Differentiation of Cell Lines:

In some embodiments, the methods described herein further includeinducing differentiation of the cell line with the genetic variation ofinterest. Differentiation of cells is accomplished by exposing cells tocharacterized factors which are known to produce a specific lineageoutcome in the cells so exposed, so as to target their differentiationto that of a specific, desired lineage and/or cell type of interest.Cells which are terminally differentiated display phenotypiccharacteristics of specialization and often lose the capacity to undergoindefinite culturing, exhibiting slowed proliferation.

The iPSCs as described herein may be differentiated into various celltypes including any cell type of interest including endodermal (givingrise to cells of the endoderm, which gives rise to inner tissues andorgans such as the alimentary canal, gut, digestive glands, respiratorysystem, and intestines/bladder), ectodermal (giving rise to cells of theectoderm, which gives rise to the nervous system, skin, and otheroutermost specialized tissues and organs), mesodermal (giving rise tocells of the mesoderm; mesoderm is the middle germ layer of an embryocoming from the inner cell mass of the blastocyst; it gives rise tobone, muscle, connective tissues, including the dermis, the bloodvascular system, the urogenital system except the bladder, andcontributes to some glands), neuroectodermal (giving rise to any cellsof the neuroectoderm, which gives rise to neurons, supporting cells, andependyma of the central nervous system and the neural crest cells thatform peripheral ganglia and a wide variety of other tissues), neural(giving rise to any cells of the nervous system peripheral and central;autonomic and somatic, including all neurons, support cells/glia, etc).In some embodiments, the cell line is differentiated into a populationof cells, for example, a cobblestone-like cell line, a cardiomyocyticcell population, an epithelial cell population such as akeratin-containing or gut-like epithelial cell population, agastrointestinal cell population, a respiratory cell population, ahepatic cell population, a pancreatic cell population, an endocriniccell population, an epidermal cell population, a myogenic cellpopulation, a cartilage cell population, a mucosal cell population, askeletal cell population, a cartilage cell population, a nephritic cellpopulation, a lymphatic cell population, a splenic cell population, orthe precursors of any of the preceding.

In some embodiments, the cell population derived from the iPSCs is amultipotent cell population. In some embodiments, the cell populationderived from the iPSCs is a monopotent cell population. In someembodiments, the cell population derived from the iPSCs is a terminallydifferentiated cell population. In some embodiments, the cell populationderived from the iPSCs is capable of undergoing passage in culturewithout observed replicative crisis, up to and including days, weeks,months and years of passage in cell culture. In some embodiments, thecell population derived from the iPSCs is incapable of undergoingpassage in culture without observed replicative crisis. In each case,the ordinarily skilled artisan can readily assess the viability andlineage potency of the derived cell population using methods known inthe art.

In certain embodiments, the cell line is differentiated into aneuroectodermal, neuronal, neuroendocrine, dopaminergic, cholinergic,serotonergic (5-HT), glutamatergic, GABAergic, adrenergic,noradrenergic, sympathetic neuronal, parasympathetic neuronal,sympathetic peripheral neuronal, or glial cell population, such as amicroglial (amoeboid, ramified, activated phagocytic, activatednon-phagocytic) or macroglial (central nervous system (CNS): astrocytes,oligodendrocytes, ependymal cells, radial glia; peripheral nervoussystem (PNS): Schwann cells, satellite cells) cell population, or theprecursors of any of the preceding, including neural stem and progenitorcells.

Characterization of Cell Lines:

In some embodiments, the methods further include phenotypiccharacterization of the cell lines produced by the present methods, orof their progeny. For example, cells obtained from a subject with agenetic variation of interest and induced to pluripotency according tothe present methods may be characterized, prior to any further use, fortheir capacity to support teratoma formation in a murine model as aconfirmation of pluripotency. Following differentiation, the resultingprogeny cells may be, for example, stained for markers ofdifferentiation and subject to a teratoma formation assay to demonstratecompletion of differentiation. The phenotypic characterization cancomprise exposing the cell line to an agent or condition and observingany change or lack of change in the cell line.

Prior to and following differentiation of the subject iPSCs, thephenotypic characterization frequently comprises staining for one ormore markers according to methods known in the art and discussed above.In many embodiments, the one or more markers are lineage markers such asMap2, type III beta tubulin, doublecortin, NeuN, glial fibrillary acidicprotein, S100-beta, NG2, GalC, tyrosine hydroxylase, aromatic amino aciddecarboxylase, Grk2, glutamate transporter, GAD, dopamine betahydroxylase, cellular uptake system molecules, etc. See, e.g. Peng etal., supra. Lineage markers corresponding to other terminallydifferentiated neural subtypes, as well as to stem, progenitor and othercells, are known in the art and may be used as well.

In some embodiments, the cell derived from iPSCs according to thepresent methods is a neural cell; in certain embodiments, the cell lineis differentiated into a neuroectodermal cell, neuronal cell,neuroendocrine cell, dopaminergic cell, cholinergic cell, serotonergic(5-HT) cell, glutamatergic cell, GABAergic cell, adrenergic cell,noradrenergic cell, sympathetic neuronal cell, parasympathetic neuronalcell, sympathetic peripheral neuronal cell, or glial cell, such as amicroglial (amoeboid, ramified, activated phagocytic, activatednon-phagocytic) or macroglial (CNS: astrocytes, oligodendrocytes,ependymal cells, radial glia; PNS: Schwann cells, satellite cells) cell,or precursors of any of the preceding.

In some embodiments, the change or lack of change is observed bystaining for one or more markers of cytotoxicity, oxidative stress,cellular transport, apoptosis, mitochondrial function, ubiquitinfunction, and proteasomal function according to standard methods, asdiscussed herein. In some embodiments, the change or lack of change isobserved by testing for one or more of ATP production, LDH release,activated caspase levels, and expression of alpha-synuclein. In someembodiments, the change or lack of change is observed by one or more offlow cytometry, quantitative real-time PCR, and induction of teratomasin mice. Those of skill in the art will be able to determine how toobserve a change or lack of change with no more than routine skill.

Disorders with Genetic Variations:

In some embodiments, the subject with the genetic variation of interesthas been diagnosed with a disorder or a predisposition to a disorder.Disorders wherein the present methods and compositions find use aredescribed below, though one of skill in the art will appreciate that themethods described herein are generally applicable to all diseases anddisorders involving CNV and other genetic variations.

Parkinson's Disease:

Parkinson's disease is one of the most common neurodegenerative diseasesof aging. Approximately 1-2% of the population over 65 years is affectedby this disorder, and it is estimated that the number of prevalent casesof Parkinson's disease will double by the year 2030 (Dorsey, E. R. etal. Projected number of people with Parkinson disease in the mostpopulous nations, 2005 through 2030. Neurology 68, 384-6 (2007). Thedisease is slowly progressive with the cardinal features of rigidity,resting tremor, bradykinesia, and asymmetric onset, although it is nowbecoming apparent that the disease is widespread in the central andperipheral nervous system. Clinical manifestation of motor symptoms ofParkinson's disease starts when ˜40-60% of the dopaminergic neurons inthe substantia nigra of the midbrain have died. Neuropathologically, thekey features include loss of dopaminergic neurons in the substantianigra and other areas in the brain, and intracytoplasmic inclusionsknown as Lewy bodies, of which α-synuclein is a major component(Langston, J. W. The Parkinson's complex: parkinsonism is just the tipof the iceberg. Ann Neurol 59, 591-6 (2006).; Forman, M. S., Lee, V. M.& Trojanowski, J. Q. Nosology of Parkinson's disease: looking for theway out of a quagmire. Neuron 47, 479-82 (2005); Litvan, I. et al. Theetiopathogenesis of Parkinson disease and suggestions for futureresearch. Part II. J Neuropathol Exp Neurol 66, 329-36 (2007); Litvan,I. et al. The etiopathogenesis of Parkinson disease and suggestions forfuture research. Part I. J Neuropathol Exp Neurol 66, 251-7 (2007)).Currently there is no cure for the disease, nor is the cause of thedisease known.

There are several other conditions that have the features of Parkinson'sdisease and are interchangeably referred to as Parkinson's-like disease,Parkinson's-related disease, secondary Parkinsonism, Parkinson'ssyndrome, or atypical Parkinson's disease. These are neurologicalsyndromes that can be characterized by tremor, hypokinesia, rigidity,and postural instability. The underlying causes of Parkinson's-likedisease are numerous, and diagnosis can be complex. A wide-range ofetiologies can lead to a similar set of symptoms, including some toxins,a few metabolic diseases, and a handful of non-Parkinson's diseaseneurological conditions. A common cause is as a side effect ofmedications, mainly neuroleptic antipsychotics especially thephenothiazines (such as perphenazine and chlorpromazine), thioxanthenes(such as flupenthixol and zuclopenthixol) and butyrophenones (such ashaloperidol (Haldol)), piperazines (such as ziprasidone), and rarely,antidepressants. Other causes include but are not limited toolivopontocerebellar degeneration, progressive supranuclear palsy,corticobasal degeneration, temporo-frontal dementia; drug induced likeantipsychotics, prochlorperazine, metoclopromide; poisoning with carbonmonoxide; head trauma; and Huntington's disease Parkinsonism. In somecases α-synucleinopathies can result in Parkinson's-like disease,secondary Parkinsonism, Parkinson's syndrome, or atypical Parkinson's.

Alzheimer's Disease:

Alzheimer's disease is a progressive neurodegenerative disorder which isthe predominant cause of dementia in people over 65 years of age.Clinical symptoms of the disease generally begin with subtle short termmemory problems and as the disease progresses, difficulties with memory,language and orientation occur more frequently. In late stageAlzheimer's disease, ventricular enlargement and shrinkage of the brainmay be observed by magnetic resonance imaging. Some characteristicchanges in the Alzheimer's disease brain include neuronal loss inselected regions; intracellular neurofibrillary tangles (NFTs) in theneurons of the cerebral cortex and hippocampus; and neuritic plaquescontaining amyloids that may be further surrounded by dystrophicneuriteism reactive astrocytes and microglia. See, e.g., Wisniewski etal., Biochem. Biophys. Res. Comm. 192:359 (1993). The NFTscharacteristic of Alzheimer's disease consist of abnormal filamentsbundled together in neuronal cell bodies. The intracellularneurofibrillary tangles are composed of polymerized tau protein, andabundant extracellular fibrils are comprised largely of beta-amyloid.Beta-amyloid, also known as A_(beta), arises from the proteolyticprocessing of the amyloid precursor protein (APP) at the beta- andgamma-secretase cleavage sites giving rise to the cellular toxicity andamyloid-forming capacity of the two major forms of A_(beta) (A_(beta)40and A_(beta)42). Thus, preventing APP processing into plaque-producingforms of amyloid may critically influence the formation and progressionof the disease making BACE1 (including variants thereof, e.g. variantsA, B, C, and D) a clinical target for inhibiting or arresting thisdisease. The presenilins are additional candidate targets forredirecting aberrant processing. See, e.g., Tagami et al., NeurodegenerDis. 5(3-4):160-2 (2008). What are referred to as “Ghost” NFTs are alsoobserved in Alzheimer's disease brains, presumably marking the locationof dead neurons. Other neuropathic features of Alzheimer's diseaseinclude granulovacuolar changes, neural loss, gliosis and the variablepresence of Lewy bodies. The identification between genetic loci andneurodegenerative changes or associations between genetic loci and therisk of developing a neurodegenerative disease may be useful in methodsof diagnosing, screening and prognosing patients, as well as intherapeutic development methods. An accurate cellular model system forstudy of the disease would further these goals.

Autism Spectrum Disorders:

Autism spectrum (AS) disorders include three separate diagnoses, whichinclude autism, Asperger's syndrome and Pervasive Developmental Delay(PDD). PDD is characterized by developmental delays of sociability,communication and use of imagination. Asperger's syndrome is a moresevere form of PDD but lacks the language and intelligence deficitsnormally associated with autism. Autism is exemplified by severecommunication impairments, social interaction deficits andrepetitive/stereotypic behaviors. Each of these disorders has specificdiagnostic criteria as outlined by the American Psychiatric Association(APA) in its Diagnostic & Statistical Manual of Mental Disorders(DSM-IV-TR). Autism impacts the normal development of the brain in theareas of social interaction and communication skills Children and adultswith autism typically have difficulties in verbal and non-verbalcommunication, social interactions, and leisure or play activities. Datafrom whole-genome screens in multiplex families suggest interactions ofat least 10 genes in the causation of autism. Thus far, a putativespeech and language region at 7q31-q33 seems most strongly linked toautism, with linkages to multiple other loci. Cytogenetic abnormalitiesat the 15q11-q13 locus are fairly frequent in people with autism, and a“chromosome 15 phenotype” was described in individuals with chromosome15 duplications. Among other candidate genes are the FOXP2, RAY1/ST7,IMMP2L, and RELN genes at 7q22-q33 and the GABA(A) receptor subunit andUBE3A genes on chromosome 15q11-q13. Variant alleles of the serotonintransporter gene (5-HTT) on 17q11-q12 are more frequent in individualswith autism than in nonautistic populations. See, e.g., Muhle et al.,Pediatrics 113(5):e472-86 (2004). A variant allele of the Engrailedgene, which maps to chromosome 7, in particular, to 7q36.3, has beenimplicated in the autism spectrum disorders. See, for example, US PatentPublication No. 2006/0141519.

Rett Syndrome:

Rett syndrome (or disorder) is a disorder of brain development thatoccurs almost exclusively in girls. After 6 to 18 months of apparentlynormal development, girls with the classic form of Rett syndrome developsevere problems with language and communication, learning, coordination,and other brain functions. Early in childhood, affected girls losepurposeful use of their hands and begin making repeated hand wringing,washing, or clapping motions. They tend to grow more slowly than otherchildren and have a small head size (microcephaly). Other signs andsymptoms can include breathing abnormalities, seizures, an abnormalcurvature of the spine (scoliosis), and sleep disturbances. Researchershave described several variants of Rett syndrome with overlapping signsand symptoms. The atypical forms of this disorder range from a mildtype, in which speech is preserved, to a very severe type that has noperiod of normal development. A form of Rett syndrome called theearly-onset seizure variant has most of the characteristic features ofclassic Rett syndrome, but also causes seizures that begin in infancy.The condition affects an estimated 1 in 10,000-22,000 females.

Mutations in the CDKL5 and MECP2 genes cause Rett syndrome. Most casesof classic Rett syndrome are caused by mutations in the MECP2 (methylCpG binding protein 2) gene. This gene provides instructions for makinga protein (MeCP2) that is critical for normal brain development. TheMeCP2 protein likely plays a role in forming connections (synapses)between nerve cells. It is believed that this protein has severalfunctions, including regulating other genes in the brain by switchingthem off when they are not needed. The MeCP2 protein may also controlthe production of different versions of certain proteins in nerve cells.Although mutations in the MECP2 gene disrupt the normal function ofnerve cells, it is unclear how these mutations lead to the signs andsymptoms of Rett syndrome. See, e.g., Amir, et al., Nat. Genet.23(2):127-8 (1999). Males with mutations in the MECP2 gene often diebefore birth or in infancy. A small number of males with a MECP2mutation, however, have developed signs and symptoms similar to those ofclassic Rett syndrome. Some of these boys have an extra X chromosome inmany or all of the body's cells. The extra X chromosome contains anormal copy of the MECP2 gene, which produces enough of the MeCP2protein for the boys to survive. Other males with features of Rettsyndrome have mutations in the MECP2 gene that occur after conceptionand are present in only a fraction of the body's cells. In rare cases,the MECP2 gene is abnormally duplicated in boys with mental retardationand some developmental problems characteristic of Rett syndrome.

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene cause anatypical form of Rett syndrome in females called the early-onset seizurevariant. See, e.g., Evans et al., Eur J Hum Genet. 13(10): 1113-20(2005). The CDKL5 gene provides instructions for making a protein thatappears to be essential for normal brain development. Although thefunction of this protein is unknown, it may play a role in regulatingthe activity of other genes. The CDKL5 protein acts as a kinase. Atleast 10 mutations in the CDKL5 gene, some in the kinase domain, havebeen identified in girls with atypical form Rett syndrome. This severeform of the disorder includes many of the features of classic Rettsyndrome (including developmental problems, loss of language skills, andrepeated hand wringing or hand washing movements), but also causesrecurrent seizures beginning in infancy. How the identified defects inthe CDKL5 gene produce the symptoms remains unclear.

Lewy Body Disease:

Lewy Body disease, or Dementia with Lewy Bodies (DLB) is one of the mostcommon types of progressive dementia. The central feature of DLB isprogressive cognitive decline, combined with three additional definingfeatures: (1) pronounced “fluctuations” in alertness and attention, suchas frequent drowsiness, lethargy, lengthy periods of time spent staringinto space, or disorganized speech; (2) recurrent visual hallucinations,and (3) parkinsonian motor symptoms, such as rigidity and the loss ofspontaneous movement. Individuals may also suffer from depression. Thesymptoms of DLB are caused by the build-up of Lewy bodies, i.e.,accumulated bits of alpha-synuclein protein, inside the nuclei ofneurons in areas of the brain that control particular aspects of memoryand motor control. It is not understood why alpha-synuclein accumulatesinto Lewy bodies or how Lewy bodies cause the symptoms of DLB.Alpha-synuclein accumulation is also linked to Parkinson's disease,multiple system atrophy, and several other disorders, which are referredto as the “synucleinopathies.” DLB usually occurs sporadically, inpeople with no known family history of the disease. However, rarefamilial cases have occasionally been reported. Genes associated withDLB include SNCA, encoding alpha synuclein and GBA, encodingbeta-glucocerebrosidase.

Hallervorden-Spatz Syndrome:

Pantothenate kinase-associated neurodegeneration (PKAN), also known asHallervorden-Spatz syndrome, is a degenerative disease of the brain,which can lead to the display of parkinsonism in the affectedindividual. Neurodegeneration in PKAN is accompanied by an excess ofiron that progressively builds in the brain. Symptoms typically begin inchildhood and are progressive, often resulting in death by earlyadulthood. Symptoms of PKAN begin before middle childhood, and mostoften are noticed before ten years of age. Symptoms include dystonia,dysphagia & dysarthria due to involvement of muscle groups involved inspeech, rigidity/stiffness of limbs, tremor, writhing movements,dementia, spasticity, weakness, seizures and pigmentary retinopathy,another degenerative condition that affects the individual's retina,often causing alteration of retinal color and progressive deteriorationof the retina at first causing night blindness and later resulting in acomplete loss of vision. 25% of individuals experience anuncharacteristic form of PKAN that develops post-10 years of age andfollows a slower, more gradual pace of deterioration than those pre-10years of age. These individuals face significant speech deficits as wellas psychiatric and behavioral disturbances. Being a progressive,degenerative nerve illness, Hallervorden-Spatz leads to early immobilityand often death by early adulthood.

PKAN is an autosomal recessive disorder, and those heterozygous for thedisorder may not display any atypical characteristics that areconsidered suggestive of the disorder. The disorder is caused by amutant PANK2 gene located at the chromosomal locus: 20p13-p12.3. PANK2encodes pantothenate kinase 2, which in turn is responsible forpreventing the accumulation of N-pantothenoyl-cysteine and pantetheine.It is believed that when this accumulation is not suppressed, the resultis direct cell toxicity or cell toxicity as a result of free radicaldamage due to the lack of suppression. PANK2 encodes a 1.85 Kbtranscript which is derived from seven exons covering a total distanceof approximately 3.5 Mb of genomic DNA. The PANK2 gene also encodes a50.5-kDa protein that is a functional pantothenate kinase, an essentialregulatory enzyme in coenzyme A (CoA) biosynthesis, and catalyzing thephosphorylation of pantothenate (Vitamin B5), N-pantothenoyl-cysteine,and pantetheine (OMIM). Mutant PANK2 gene coded proteins are oftencaused by null or missense mutations, most notably a 7 bp deletion inthe PANK2 gene coding sequence. See, e.g., Pellecchia et al., Thediverse phenotype and genotype of pantothenate kinase-associatedneurodegeneration, Neurology 64 (10): 1810-2 (2005).

Progressive Supranuclear Palsy:

Progressive supranuclear palsy (PSP) is a rare degenerative disorderinvolving the gradual deterioration and death of selected areas of thebrain. The sexes are affected approximately equally and there is noracial, geographical or occupational predilection. Approximately 6people per 100,000 population have PSP. The initial symptom intwo-thirds of cases is loss of balance and falls. Other common earlysymptoms are changes in personality, general slowing of movement, andvisual symptoms. Later symptoms and signs are dementia (typicallyincluding loss of inhibition and ability to organize information),slurring of speech, difficulty swallowing, and difficulty moving theeyes, particularly in the vertical direction. The latter accounts forsome of the falls experienced by these patients as they are unable tolook up or down. Some of the other signs are poor eyelid function,contracture of the facial muscles, a backward tilt of the head withstiffening of the neck muscles, sleep disruption, urinary incontinenceand constipation

Fewer than 1% of those with PSP have a family member with the samedisorder. A variant in the gene for tau protein called the H1 haplotype,located on chromosome 17, has been linked to PSP. Nearly all individualswith PSP bear a copy of that variant from each parent, but this is trueof about two-thirds of the general population. Therefore, the H1haplotype appears to be necessary but not sufficient to cause PSP. Othergenes, as well as environmental toxins, are being investigated as causalcontributors to PSP.

Wilson Disease:

Wilson disease is a rare autosomal recessive inherited disorder ofcopper metabolism. The condition is characterized by excessivedeposition of copper in the liver, brain, and other tissues. The majorphysiologic aberration is excessive absorption of copper from the smallintestine and decreased excretion of copper by the liver. The geneticdefect, localized to chromosome arm 13q, has been shown to affect thecopper-transporting adenosine triphosphatase (ATPase) gene ATP7B in theliver. Patients with Wilson disease usually present with liver diseaseduring the first decade of life or with neuropsychiatric illness duringthe third decade. The diagnosis is confirmed by measurement of serumceruloplasmin, urinary copper excretion, and hepatic copper content, aswell as the detection of Kayser-Fleischer rings. Initially, Wilsonpostulated that the familial incidence of hepatolenticular degenerationwas attributable to environmental factors rather than genetic factors.Nearly a decade later, Hall reported that Wilson disease was morefrequent in siblings. In 1953, Bearn discovered an autosomal recessivemode of inheritance confirmed by extended genetic analysis of 30families. Frydman et al localized the Wilson disease (WD) gene tochromosome 13. The WD gene product is a 1411 amino acid protein withhighest levels of expression in the liver, kidneys, and placenta. The WDgene codes for P-type copper-transporting ATPase, now characterized asATP7B. Many of the gene defects for ATP7B are small deletions,insertions, or missense mutations. Most patients carry differentmutations on each of their 2 chromosomes. More than 40 differentmutations have been identified, the most common of which is a changefrom a histidine to a glutamine (H1069Q). See, for example, Thomas, etal., The Wilson disease gene: spectrum of mutations and theirconsequences, Nat. Genet. 9(2):210-7 (1995).

Use of Induced Pluripotent Cell Lines for Screening

Methods of screening the cell lines or cell populations (iPSCs ordifferentiated iPSCs) with a variation of a gene of interest for anagent to treat a disease or disorder are also provided. The methodscomprise contacting an agent to be screened with a cell line or cellpopulation described herein, observing a change or lack of change in theone or more cells, where the change or lack of change is correlated withan ability of the agent to treat the disease or disorder. In otherwords, the change or lack of change can be indicative of an ability ofthe agent to treat the disease or disorder. Agents to be screenedinclude potential and known therapeutics. Such therapeutics include, butare not limited to, small molecules; aptamers, antisense molecules;antibodies and fragments thereof; polypeptides; proteins;polynucleotides; organic compounds; cytokines; cells; genetic agentsincluding, for example, shRNA, siRNA, a virus or genetic material in aliposome; an inorganic molecule including salts such as, for example,lithium chloride or carbonate; and the like.

In some embodiments, the methods of screening the cell lines or cellpopulations with a variation of a gene of interest for an agent to treata disease or disorder include comparison of the cell lines orpopulations with another cell line or population. For example, the celllines or cell populations described herein may be compared to a normalcell line or population, meaning a cell line derived from a patient withno known symptoms or who has not been diagnosed with the disease ordisorder of interest. Alternatively, the cell lines or cell populationsdescribed herein may be compared to a cell line or population ofidiopathic cells, meaning cell lines or populations derived frompatients who present with symptoms of the disease or disorder ofinterest, or have been diagnosed with the disease or disorder, but whodo not have a variation of the gene of interest, and where the cause ofthe disease or disorder may even be unknown (sporadic or idiopathic). Inother embodiments, the methods of screening the cell lines or cellpopulations with a variation of a gene of interest for an agent to treata disease or disorder involve comparison of the cell lines or cellpopulations derived from a cell containing a genetic variation ofinterest to both a normal cell line or cell population and an cell lineisolated from a subjecting presenting with an idiopathic/unknown form ofdisease or population. In some embodiments, the normal cell line or cellpopulation and the idiopathic cell line or population will have beengenerated using the same protocol as that used to generate the cell lineor population containing the genetic variation of interest. Thus, thenormal cell line or cell population may serve as a control. As well, anychange or lack of change in the control cells, idiopathic cells, andcells with the genetic variation of interest upon contacting with anagent may be compared to one another. Patients or groups of patientswith idiopathic disease may thereby be compared to patients with geneticvariations of interest with respect to their responsiveness to an agent,to a class of agent, to an amount of agent, and the like. In this way,idiopathic diseases are classified by their responsiveness to agents,yielding information about the etiology of the idiopathic disease and,alternatively or additionally, agents are identified which are effectiveacross one or more classes of disease. It is envisioned that thesemethods are additionally used to develop treatment regimens for patientsor classes of patients with a disease.

In other embodiments cell lines are created from patients presentingwith an idiopathic form of disease and such cell lines are used forscreening, and identification of disease mechanisms or diseasediagnosis, independent of cells lines in which genetic variations exist.

In some embodiments, the cell lines or cell populations are screened bystaining for a marker and observing a change. Nonlimiting examples of achange or lack of change include a change or lack of change in cellviability, cellular chemistry, cellular function, mitochondrialfunction, cell aggregation, cell morphology, cellular proteinaggregation, gene expression, cellular secretion, or cellular uptake.Cell stains are known to those of skill in the art. Nonlimiting examplesinclude markers of general cytotoxicity in cell viability assays,markers of apoptosis, markers of oxidative stress, markers ofmitochondrial function, and combinations thereof. Alternatively, oradditionally, screening may be effected by testing for one or more ofATP production, LDH release, activated caspase levels, expression of thegene of interest. In certain embodiments, the screening is forexpression of the gene of interest α-synuclein. See, e.g., Andreotti, P.E. et al. Chemosensitivity testing of human tumors using a microplateadenosine triphosphate luminescence assay: Clinical correlation forcisplatin resistance of ovarian carcinoma. Cancer Res. 55, 5276-82(1995); Beckers, B. et al. Application of intracellular ATPdetermination in lymphocytes for HLA typing. J. Biolumin. Chemilumin. 1,47-51 (1986); Crouch, S. P. M. et al. The use of ATP bioluminescence asa measure of cell proliferation and cytotoxicity. J. Immunol. Meth. 160,81-8 (1993); O'Brien, J. et al. Investigation of the alamar blue(resazurin) fluorescent dye for the assessment of mammalian cellcytotoxicity. Eur. J. Biochem. 267, 5421-6 (2000); Riss, T. and Moravec,R. A. Use of multiple assay endpoints to investigate effects ofincubation time, dose of toxin and plating density in cell-basedcytotoxicity assays. Assay Drug Dev. Technol. 2, 51-62 (2004).

The cells of the present method may be used for screening biologicalresponse modifiers, i.e., compounds and factors that affect the varioussignaling pathways, including pathways in dopaminergic neuronal cells. Awide variety of assays may be used for this purpose, includingimmunoassays for protein production, amount, secretion or binding;determination of cell growth, differentiation and functional activity;production of hormones; production of neurotransmitters; production ofneurohormones; measurement of reactive oxygen species and/or freeradical-mediated damage; and the like. See, e.g., Filipov et al.,Toxicology 232(1-2):68-78 (2007); Peng et al., J. Neurosci.26(45):11644-51 (2006); Yan et al., Analysis of oxidative modificationof proteins. Curr Protoc Cell Biol., Chapter 7:Unit 7.9 (2002);Armstrong et al., Measurement of Reactive Oxygen Species in Cells andMitochondria, Methods in Cell Biology, Vol 80, Chapter 18 (2007).

For example, the subject cells may be used to screen for agents thatenhance or inhibit apoptosis, or the expression of α-synuclein.Typically the candidate agent will be added to the cells, and theresponse of the cells monitored through evaluation of cell surfacephenotype, functional activity, patterns of gene expression,physiological changes, electrophysiological changes and the like. Insome embodiments, screening assays are used to identify agents that havea low toxicity in human cells.

The term “agent” as used herein describes any molecule, e.g., nucleicacid, protein or pharmaceutical, with the capability of affecting achange in a parameter of interest in the cells of the assay. Generally aplurality of assay mixtures are run in parallel with different agentconditions and/or concentrations to obtain a differential response tothe various concentrations. Typically, one of these conditions serves asa negative control, i.e., at zero concentration or below the level ofdetection. Screening may be directed to known pharmacologically activecompounds and chemical analogs thereof.

Candidate agents encompass numerous chemical classes, including organicmolecules. Candidate agents may comprise functional groups necessary forstructural interaction with proteins, such as hydrogen bonding, and mayinclude at least one amine, carbonyl, hydroxyl or carboxyl group. Incertain embodiments, the candidate agents have at least two of thefunctional chemical groups. The candidate agents may comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more functional groups. Candidateagents may also be found among biomolecules including, but not limitedto: peptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof.

Candidate agents may be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g., magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would often be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g., albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components is added in any order that provides for binding,delivery or effect. Incubations are performed at any suitabletemperature, typically ranging from 4 to 40° C., but may be higher orlower than these temperatures. Incubation periods may be selected foroptimum activity, but may also be optimized to facilitate rapidhigh-throughput screening.

Detection of change or lack of change in the cells may utilize stainingof cells, performed in accordance with conventional methods. Theantibodies of interest are added to the cell sample, and incubated for aperiod of time sufficient to allow binding to the epitope, for example,at least about 10 minutes. The antibody may be labeled with a label, forexample, chosen from radioisotopes, enzymes, fluorescers,chemiluminescers, or other labels for direct detection. Alternatively, asecond stage antibody or reagent is used to amplify the signal. Suchreagents are well known in the art. For example, the primary antibodymay be conjugated to biotin, with horseradish peroxidase-conjugatedavidin added as a second stage reagent. Final detection uses a substratethat undergoes a color change in the presence of the peroxidase. Theabsence or presence of antibody binding may be determined by variousmethods, including flow cytometry of dissociated cells, microscopy,radiography, scintillation counting, etc. One exemplary use of stainingin the present methods is described below in more detail.

Cellular gene expression may be assessed following a candidate treatmentor experimental manipulation. The expressed set of genes may be comparedwith control cells of interest, e.g., cells also derived according tothe present methods but which have not been contacted with the agent.Any suitable qualitative or quantitative methods known in the art fordetecting specific mRNAs can be used. mRNA can be detected by, forexample, hybridization to a microarray, in situ hybridization in tissuesections, by reverse transcriptase-PCR, or in Northern blots containingpoly A+ mRNA. One of skill in the art can readily use these methods todetermine differences in the size or amount of mRNA transcripts betweentwo samples. For example, the level of particular mRNAs in cellscontacted with agent is compared with the expression of the mRNAs in acontrol sample.

mRNA expression levels in a sample can be determined by generation of alibrary of expressed sequence tags (ESTs) from a sample. Enumeration ofthe relative representation of ESTs within the library can be used toapproximate the relative representation of a gene transcript within thestarting sample. The results of EST analysis of a test sample may thenbe compared to EST analysis of a reference sample to determine therelative expression levels of a selected polynucleotide.

Alternatively, gene expression in a test sample may be performed usingserial analysis of gene expression (SAGE) methodology (Velculescu etal., Science (1995) 270:484). In short, SAGE involves the isolation ofshort unique sequence tags from a specific location within eachtranscript. The sequence tags are concatenated, cloned, and sequenced.The frequency of particular transcripts within the starting sample isreflected by the number of times the associated sequence tag isencountered with the sequence population.

Gene expression in a test sample may also be analyzed using differentialdisplay (DD) methodology. In DD, fragments defined by specific sequencedelimiters (e.g., restriction enzyme sites) are used as uniqueidentifiers of genes, coupled with information about fragment length orfragment location within the expressed gene. The relative representationof an expressed gene with a sample can then be estimated based on therelative representation of the fragment associated with that gene withinthe pool of all possible fragments. Methods and compositions forcarrying out DD are well known in the art, see, e.g., U.S. Pat. Nos.5,776,683 and 5,807,680.

Alternatively, gene expression in a sample may be assessed usinghybridization analysis, which is based on the specificity of nucleotideinteractions. Oligonucleotides or cDNA can be used to selectivelyidentify or capture the DNA or RNA of specific sequence composition, andthe amount of RNA or cDNA hybridized to a known capture sequencedetermined qualitatively or quantitatively, to provide information aboutthe relative representation of a particular RNA message within the poolof cellular RNA messages in a sample. Hybridization analysis may bedesigned to allow for concurrent screening of the relative expression ofhundreds to thousands of genes by using, for example, array-basedtechnologies having high density formats, including filters, microscopeslides, or microchips, or solution-based technologies that usespectroscopic analysis (e.g., mass spectrometry).

In another screening method, the test sample is assayed at the proteinlevel. Methods of analysis may include 2-dimensional gels; massspectroscopy; analysis of specific cell fraction, e.g., lysosomes; andother proteomics approaches. For example, detection may utilize stainingof cells or histological sections (e.g., from a biopsy sample) withlabeled antibodies, performed in accordance with conventional methods.Cells can be permeabilized to stain cytoplasmic molecules. In general,antibodies that specifically bind a differentially expressed polypeptideare added to a sample, and incubated for a period of time sufficient toallow binding to the epitope, usually at least about 10 minutes. Theantibody can be detectably labeled for direct detection (e.g., usingradioisotopes, enzymes, fluorescers, chemiluminescers, and the like), orcan be used in conjunction with a second stage antibody or reagent todetect binding (e.g., biotin with horseradish peroxidase-conjugatedavidin, a secondary antibody conjugated to a fluorescent compound, e.g.,fluorescein, rhodamine, Texas red, etc.). The presence or absence ofantibody binding may be determined by various methods, including flowcytometry of dissociated cells, microscopy, radiography, scintillationcounting, etc. Any suitable alternative methods can of qualitative orquantitative detection of levels or amounts of differentially expressedpolypeptide can be used, for example ELISA, western blot,immunoprecipitation, radioimmunoassay, etc.

Conditioned media, i.e., media in which cells of the methods describedherein have been grown for a period of time sufficient to allowsecretion of soluble factors into the culture, may be isolated atvarious stages and the components analyzed for the presence of factorssecreted by the cells. Separation can be achieved with HPLC, reversedphase-HPLC, gel electrophoresis, isoelectric focusing, dialysis, orother non-degradative techniques, which allow for separation bymolecular weight, molecular volume, charge, combinations thereof, or thelike. One or more of these techniques may be combined to enrich furtherfor specific fractions.

Use of Induced Pluripotent Cell Lines to Elucidate Disease Progressionand Mechanism

In some embodiments, the cell lines and cell populations (iPSCs ordifferentiated iPSCs) described herein are used to study the mechanismof a disease of interest. In such embodiments, a molecular determinantof a disorder of interest is identified by contacting one or more testcells from a cell line derived by the method as described herein with anagent or condition which affects a pathway of interest, such as acellular pathway, such as a disease-associated gene pathway, andobserving any change or lack of change in the one or more test cells. Adisease-associated gene pathway generally refers to genes and geneproducts comprising a disease-associated gene, and may include one ormore genes that act upstream or downstream of a disease-associated genein a disease related pathway; or any gene whose gene product interactswith, binds to, competes with, induces, enhances or inhibits, directlyor indirectly, the expression or activity of a disease-associated gene;or any gene whose expression or activity is induced, enhanced orinhibited, directly or indirectly, by a disease-associated gene; or anygene whose gene product is induced, enhanced or inhibited, directly orindirectly, by a disease-associated gene. A disease-associated genepathway may refer to one or more genes or the gene products which act ina signaling pathway. Direct and indirect mechanisms refer, respectively,to direct contact or modification of a molecular actor in a pathway andcontact or modification of an intermediary molecule which in turncontacts or modifies a molecular actor in a pathway, as is known in theart. Indirect mechanisms may be one or more steps removed from directinfluence on a pathway. “Molecular determinants,” as used herein, refersto any of the genes or gene products which may act, directly orindirectly, in a disease-associated gene pathway.

In some embodiments, the test cells are compared to one or more controlcells. In some of these embodiments, such control cells are cells of thetest cell line that have not been contacted with the agent or conditionas described above. In further embodiments, such control cells are froma second cell line derived from a cell type which is the same as that ofthe test cell line with the exception that it lacks the geneticvariation of interest; i.e., the second cell line is produced byinducing dedifferentiation according to the same method used todedifferentiate the test cell line; and the resulting control cell lineis contacted with the same agent or condition as the test cell lineduring experimentation.

In the methods employing genetic agents, polynucleotides may be added tothe cells in order to alter the genetic composition of the cells. Outputparameters are monitored to determine whether there is a change inphenotype affecting particular pathways in cells derived from iPS celllines obtained from subjects diagnosed with a disease relative to thosewithout. In this way, genetic sequences may be identified that encode oraffect expression of proteins in pathways of interest, particularlypathways associated with aberrant physiological states such as thedisease of interest.

In some embodiments of the present method, agents of interest may benaturally occurring compounds such as, e.g., known compounds that havesurface membrane receptors and induce a cellular signal that results ina modified phenotype, or synthetic compounds that mimic the naturallyoccurring agents. In some embodiments, agents of interest includeinorganic compounds such as salts which are known to affect a particularcellular pathway or pathways. In some instances, the agents will actintracellularly by passing through the cell surface membrane andentering the cytosol with binding to components in the cytosol, nucleusor other organelle.

In some embodiments, the cells are subjected to a condition, whichtriggers the activities of known factors in response to the condition,using the activity of the naturally occurring factors to therebyidentify pathways and molecules associated with the disease of interest.Such conditions include, for example, hypoxic or anoxic conditions orany condition resulting in oxidative, endoplasmic reticular ormitochondrial stress.

For assays with genetic agents, the same approach may be used. Thegenetic agents are added to cells, which may be derived from iPSobtained from a subject diagnosed with a disorder of interest, e.g., adisease. Parameters associated with the pathways related to the diseasestate are monitored. Where the parameters show a pattern indicating theup or down regulation of a pathway, the agent or condition is deduced toencode or affect the expression of a member of the pathway that has aneffect on the disease state. In this way one can determine the role agene plays in the physiological state of interest, as well as definetargets for therapeutic application.

In some embodiments of the methods described herein, the change or lackof change in the cells is observed by staining, according to knownmethods. The staining may be for one or more markers, for example, oneor more markers of cytotoxicity, oxidative stress, cellular transport,apoptosis, mitochondrial function, ubiquitin function, lysosomalfunction and proteasomal function. The change or lack of change may beobserved by testing for one or more of ATP production, LDH release,activated caspase levels, and expression of alpha-synuclein according tomethods as described. The change or lack of change in cells is observedby one or more of, for example, flow cytometry, quantitative real-timePCR, and induction of teratomas in mice.

Use of Induced Pluripotent Cell Lines for Diagnostics, Prognostics, andTheranostics

Also provided herein are methods of determining the progression of adisorder and methods of determining the rate of progression of adisorder, useful for disease diagnosis, prognosis, and theranosis. Themethods includes producing an iPS cell line from an individual diagnosedwith the disorder according to the methods described herein, observingany change or lack of change in the cell line or progeny thereof,correlating the change or lack of change with progression and/or rate ofprogression, obtaining a tissue sample from a control subject andobserving the change or lack of change in the tissue sample from thesubject, where the change or lack of change is indicative of theprogression of a disorder and/or rate of disease progression. Thesubject methods use an observed characteristic of an iPS, or its progenyderived from an individual diagnosed with the disorder, as a way ofdetermining the progression and/or rate of progression of the disorderin, e.g., a second individual. The methods may further includecontacting the iPS cell or its progeny with an agent or condition andobserving the change or lack of change. In the subject methods, thechange or lack of change is indicative of the progression and/or rate ofprogression of the disorder. The change or lack of change can be achange in one or more of morphology, gene expression, protein synthesis,and secretion of gene products from the cell line, as well as otherchanges known to those of skill in the art. This change or lack ofchange in the iPS cell or progeny obtained from a first individual maybe correlated with disease progression and/or rate of diseaseprogression by, for example, detecting relative amounts of the change orlack of change in tissue samples (for example, a primary tissue sample)from individuals with early and late stage progression of the disorderor slow-progressing and fast-progressing disorders. Once thiscorrelation has been observed, the subject change or lack of change maybe used as a signature to determine the stage, i.e., progression, of thedisorder in the same or in another individual diagnosed with thedisorder. Alternatively, or in addition, the change or lack of changemay be used as a signature to determine the rate of progression of thedisorder in the same or in another individual diagnosed with thedisorder. For example, patients diagnosed with a disorder may be furtherclassified as having a slow-progressing or a fast-progressing disorder.In this way, a characteristic of an iPS cell line or its progeny derivedaccording to the methods herein is used to assess the progression orrate of progression of a disorder, the progression or rate thereof isdirectly or indirectly related to the characteristic.

Methods of Treatment

Methods of treating and/or preventing a disorder (e.g., disease) in asubject in need thereof are provided herein. The methods involveadministering to the subject an agent, e.g., identified by the screeningmethods described herein, in an effective amount to treat or prevent thedisorder.

Any disorder associated with CNV or any other genetic variation ormutation may be treated by the methods provided herein. Such disordersinclude, but are not limited to, Parkinson's disease, a toxicity-inducedParkinsonism, Alzheimer's disease, dementia, an autism spectrumdisorder, susceptibility to viral infection such as HIV, and coloboma ofthe eye, heart defects, atresia of the choanae, retardation of growthand/or development, Genital and/or urinary abnormalities, and Earabnormalities and deafness (CHARGE) syndrome. Autism spectrum disordersinclude Asperger syndrome, autism, PDD not otherwise specified, and Rettdisorder.

Other known disorders related to CNV treatable by the methods describedherein include, but are not limited to, 12q14 microdeletion syndrome,15q13.3 microdeletion syndrome, 15q24 recurrent microdeletion syndrome,16p11.2-p12.2 microdeletion syndrome, 17q21.3 microdeletion syndrome,1p36 microdeletion syndrome, 1q21.1 recurrent microdeletion, 1q21.1recurrent microduplication, 1q21.1 susceptibility locus forThrombocytopenia-Absent Radius (TAR) syndrome, 22q11 deletion syndrome(Velocardiofacial/DiGeorge syndrome), 22q11 duplication syndrome,22q11.2 distal deletion syndrome, 22q13 deletion syndrome(Phelan-Mcdermid syndrome), 2p15-16.1 microdeletion syndrome, 2q33.1deletion syndrome, 2q37 monosomy, 3q29 microdeletion syndrome, 3q29microduplication syndrome, 6p deletion syndrome, 7q11.23 duplicationsyndrome, 8p23.1 deletion syndrome, 9q subtelomeric deletion syndrome,Adult-onset autosomal dominant leukodystrophy (ADLD), Angelman syndrome(Type 1), Angelman syndrome (Type 2), ATR-16 syndrome, AZFa, AZFb,AZFb+AZFc, AZFc, Cat-Eye Syndrome (Type I), Charcot-Marie-Tooth syndrometype 1A (CMT1A), Cri du Chat Syndrome (5p deletion), Early-onsetAlzheimer disease with cerebral amyloid angiopathy, Familial AdenomatousPolyposis, Hereditary Liability to Pressure Palsies (HNPP), Leri-Weilldyschondrostosis (LWD)-SHOX deletion, Miller-Dieker syndrome (MDS),NF1-microdeletion syndrome, Pelizaeus-Merzbacher disease, Potocki-Lupskisyndrome (17p11.2 duplication syndrome), Potocki-Shaffer syndrome,Prader-Willi syndrome (Type 1), Prader-Willi Syndrome (Type 2), RCAD(renal cysts and diabetes), Rubinstein-Taybi Syndrome, Smith-MagenisSyndrome, Sotos syndrome, Split hand/foot malformation 1 (SHFM1),Steroid sulphatase deficiency (STS), WAGR 11p13 deletion syndrome,Williams-Beuren Syndrome (WBS), Wolf-Hirschhorn Syndrome, and Xq28(MECP2) duplication.

In some embodiments, a subject with the genetic variation of interesthas been diagnosed with a disorder or predisposition to a disorderassociated with protein aggregation. Such disorders include, but are notlimited to, Alzheimer's disease, Parkinson's disease, dementia, autismspectrum disorders, susceptibility to viral infection, diffuse Lewy bodydisease or any other Lewy body disorder or synucleinopathy, corticobasaldegeneration, encephalitis lethargica, multiple system atrophy,pantothenate kinase-associated neurodegeneration (Hallervorden-Spatzsyndrome), progressive supranuclear palsy, vascular Parkinsonism, Wilsondisease, hereditary pancreatitis, glomerulonephritis, human systemiclupus erythematosus, paraneoplastic syndrome, frontotemporal dementiawith Parkinsonism chromosome 17, Huntington's disease, spinocerebellarataxias, amytropic lateral sclerosis, and Creutzfeldt-Jakob disease.

In some embodiments, the disorder is Parkinson's disease (PD) or aPD-related disease. PD-related diseases include diseases, conditions,symptoms or susceptibilities to diseases, conditions or symptoms, thatinvolve, directly or indirectly, neurodegeneration including but notlimited to the following: Alzheimer's disease, amyotrophic lateralsclerosis (ALS), Alpers' disease, Batten disease, Cockayne syndrome,corticobasal ganglionic degeneration, Huntington's disease, Lewy bodydisease, Pick's disease, motor neuron disease, multiple system atrophy,olivopontocerebellar atrophy, Parkinson's disease, postpoliomyelitissyndrome, prion diseases, progressive supranuclear palsy, Rett syndrome,Shy-Drager syndrome and tuberous sclerosis. Certain PD-related diseasesare neurodegenerative diseases that affect neurons in the brain. APD-related disease may be e.g. a condition that is a risk factor fordeveloping PD, or may be a condition for which PD is a risk factor, orboth.

The agents described herein can be administered in a variety ofdifferent ways. The therapeutic agents, identified by the screeningmethods described herein, may be incorporated into a variety offormulations for therapeutic administration by combination withappropriate pharmaceutically acceptable carriers or diluents, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration of the compounds may be achieved invarious ways, including intracranial, oral, buccal, rectal, parenteral,intraperitoneal, intravenous, intramuscular, topical, subcutaneous,subdermal, intradermal, transdermal, intrathecal, nasal, intracheal,etc., administration. The active agent may be systemic afteradministration or may be localized by the use of regionaladministration, intramural administration, or use of an implant thatacts to retain the active dose at the site of implantation. For example,the agent may be intracranially administered using, e.g., an osmoticpump and microcatheter or other neurosurgical device to delivertherapeutic agents to selected regions of the brain under singular,repeated or chronic delivery regimens. In some embodiments, an agent cancross and or even readily pass through the blood-brain barrier, whichpermits, e.g., oral, parenteral or intravenous administration.Alternatively, the agent can be modified or otherwise altered so that itcan cross or be transported across the blood brain barrier. Manystrategies known in the art are available for molecules crossing theblood-brain barrier, including but not limited to, increasing thehydrophobic nature of a molecule; introducing the molecule as aconjugate to a carrier, such as transferring, targeted to a receptor inthe blood-brain barrier, or to docosahexaenoic acid etc. In anotherembodiment, an agent is administered via the standard procedure ofdrilling a small hole in the skull to administer the agent. In anotherembodiment, the molecule can be administered intracranially or, forexample, intraventricularly. In another embodiment, osmotic disruptionof the blood-brain barrier can be used to effect delivery of agent tothe brain (Nilayer et al., Proc. Natl. Acad. Sci. USA 92:9829-9833(1995)). In yet another embodiment, an agent can be administered in aliposome targeted to the blood-brain barrier. Administration ofpharmaceutical agents in liposomes is known (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofinfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. pp. 317-327 and 353-365 (1989). All of such methods areenvisioned herein.

Therapeutic agents may include, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers of diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, buffered water, physiologicalsaline, PBS, Ringer's solution, dextrose solution, and Hank's solution.In addition, the pharmaceutical composition or formulation may includeother carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenicstabilizers, excipients and the like. The compositions may also includeadditional substances to approximate physiological conditions, such aspH adjusting and buffering agents, toxicity adjusting agents, wettingagents, and detergents.

Further guidance regarding formulations that are suitable for varioustypes of administration may be found in Remington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).For a brief review of methods for drug delivery, see, Langer, Science249:1527-1533 (1990).

In one embodiment, the agents are useful for modulating α-synuclein.Agents that can modulate α-synuclein can be selected from but notlimited to those presented in Table 1. In certain embodiments modulationcan include but not be limited to altered fibrillation, folding,ubiquitination, trafficking, synaptic targeting, lysosomal storage,expression, subcellular compartmentalization, and lipid-interactions.

TABLE 1 Compounds that modulate α-synuclein function apomorphinecefamandole sodium fisetin pyrogallol cephaloridine luteolin1,4-naphthoquinone myricetin fustin cisplatin 6,2′,3′-trihydroxyflavoneepicatechin gallate isoproterenol 5,7,3′,4′,5′- catechinpentahydroxyflavone pyrogallin 7,3′,4′,5′-tetrahydroxyflavone alizarincianidanol (5,6,7,4′-tetrahydroxyflavone) tannic acid sulfasalazinebaicalein eriodyctol quinalizarin eriodictyol carboplatin benserazide7,3′,4′-trihydroxyisoflavone purpurogallin-4-carboxylic acidhexachlorophene epigallocatechin gallate koparin pyrvinium pamoatequercetin 2,3,4-trihydroxy-4′- ethexybenzophenone dobutamine gossypetin(3,5,7,8,3′,4′- baeomycesic acid hexahydroxyflavone) methyl-dopa2′,3′-dihydroxyflavone hamtoxylin curcumin 3′,4′-dihydroxyflavoneiriginol hexaaceatate berberine chloride 5,6-dihydroxy-7-4-acetoxyphenol methoxyflavone daidzein baicalein-7-methyl ethertheaflavin monogallate merbromin Levodopa (L-Dopa) theaflavin digallatenorepinephrine DOPAC stictic acid dopamine hydrochloride homogentisicacid purpurogallin carbidopa 6-hydroxydopamine2,5-dihydroxy-3,4-dimethoxy-4′- ethoxybenzophenone ethylnorepinephrinehydrochloride epinephrine promethazine hydrochloride tannic acid3,4-dihydroxycinnamic acid oxidopamine hydrochlorideelaidyphosphocholine 2,3-dihydroxynaphthalene pyrantel pamoatehydroquinone 3,4-dihydroxybenzoic acid elaidylphosphocholinechlorophyllide Cu complex Na salt 3,4,5-trihydroxybenzoic acidamphotericin B methyldopa 1,2,3-trihydroxybenzoic acid gallic acidisoproterenol hydrochloride gallate (gallic acid) fumarprotocetraricacid benserazide hydrochloride benzoquinone theaflavin dopamine catecholhaematoxylin pentaacetate dobutamine hydrochloride rifampicin4-methoxydalbergione thyroid hormone rosmarinic acidepigallocatechin-3-monogallate purpurin baicalin rolitetracycline Sodiumbeta-nicotinamide adenine tanshinones I and II 7,3′-dimethoxyflavonedinucleotide phosphate lansoprazole emodin liquiritigenin dimethyl etherdyclonine hydrochloride procyanidin B4 catechin pentaacetate pramoxinehydrochloride resveratrol apigenin azobenzene rutin3,4-dedesmethyl-5-deshydroxy-3′- ethoxyscleroin

The agents identified by the subject methods can be administered forprophylactic and/or therapeutic treatments. Toxicity and therapeuticefficacy of the active ingredient may be determined according tostandard pharmaceutical procedures in cell cultures and/or experimentalanimals, including, for example, determining the LD50 (the dose lethalto 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD50/ED50. Compounds that exhibit large therapeutic indicesare used in some embodiments.

The data obtained from cell culture and/or animal studies may be used informulating a range of dosages for humans. The dosage of the activeingredient typically lines within a range of circulating concentrationsthat include the ED50 with low toxicity. The dosage may vary within thisrange depending upon the dosage form employed and the route ofadministration utilized.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

The effective amount of a therapeutic agent to be given to a particularpatient will depend on a variety of factors, several of which will bedifferent from patient to patient. A competent clinician will be able todetermine an effective amount of a therapeutic agent to administer to apatient. Dosage of the agent will depend on the treatment, route ofadministration, the nature of the therapeutics, sensitivity of thepatient to the therapeutics, etc. Utilizing LD50 animal data, and otherinformation, a clinician can determine the maximum safe dose for anindividual, depending on the route of administration. Utilizing ordinaryskill, the competent clinician will be able to optimize the dosage of aparticular therapeutic composition in the course of routine clinicaltrials. The compositions can be administered to the subject in a seriesof more than one administration. For therapeutic compositions, regularperiodic administration will sometimes be required, or may be desirable.Therapeutic regimens will vary with the agent, e.g., some agents may betaken for extended periods of time on a daily or semi-daily basis, whilemore selective agents may be administered for more defined time courses,e.g., one, two three or more days, one or more weeks, one or moremonths, etc., taken daily, semi-daily, semi-weekly, weekly, etc.

A pharmaceutically or therapeutically effective amount of the agent isdelivered to the subject. The precise effective amount will vary fromsubject to subject and will depend upon the species, age, the subject'ssize and health, the nature and extent of the condition being treated,recommendations of the treating physician, and the therapeutics orcombination of therapeutics selected for administration. Thus, theeffective amount for a given situation can be determined by routineexperimentation. For purposes of the present method, generally atherapeutic amount may be in the range of about 0.001 mg/kg to about 100mg/kg body weight, in at least one dose. The subject may be administeredin as many doses as is required to reduce and/or alleviate the signs,symptoms, or causes of the disorder in question, or bring about anyother desired alteration of a biological system.

Creation of Cell Lines from Induced Pluripotent Stem Cells Specific toParkinson's Disease and Parkinson's-Like Disease

Overview:

Because most available therapies for Parkinson's disease or relatedParkinson's-like diseases are only symptomatic and lead to dose-limitingside-effects over time, there is a great need for the development of newand safe therapies to halt or even reverse disease progression.

The methods and cell lines disclosed herein are now described withrespect to Parkinson's disease and Parkinson's-like disease, though oneof skill in the art will appreciate that the methods of generating andscreening cell lines described herein are generally applicable to alldiseases and disorders involving CNV and other genetic variations.

To date, several different genes have been identified that are relatedto Parkinson's disease (Farrer, M. J. Genetics of Parkinson disease:paradigm shifts and future prospects. Nat Rev Genet. 7, 306-18 (2006).Several of these have provided major insights into proteins and pathwaysthat are likely to be important for sporadic Parkinson's disease. Afundamental insightful observation came from the discovery thatoverexpression of the normal SNCA gene in familial cases of Parkinson'sdisease due to SNCA duplications or triplications leads to many featuresof PD (Singleton, A. B. et al. alpha-Synuclein locus triplication causesParkinson's disease. Science 302, 841 (2003); Chartier-Harlin, M. C. etal. Alpha-synuclein locus duplication as a cause of familial Parkinson'sdisease. Lancet 364, 1167-9 (2004)). In humans, normal α-synuclein, whenoverexpressed can induce many of the clinical and pathological featuresof Parkinson's disease.

Using the methods described herein, human iPSCs may be generated frompatients with Parkinson's disease, for example due to a triplication ofthe SNCA gene or due to a mutation in the LRRK2 gene or due to any othergenetic variation/mutation relevant to Parkinson's disease or relatedParkinson's-like diseases. The iPSCs are differentiated into cell typesof interest known to be affected in Parkinson's disease or Parkinson'sdisease. These cells can be central or peripheral cells; can be neuronalor glial; can be autonomic or sympathetic; or can be for exampledopaminergic neurons, serotonergic neurons, cholinergic neurons,GABAergic neurons, glutamatergic neurons, or peptidergic, neurons.

Generation of a Bank of iPSCs from Humans with Parkinson's Disease orParkinson's Related Disease:

In some embodiments, the first step will be to create and expand a cellbank of iPSC lines from adult patients with specific genetic forms ofParkinson's disease, from adult patients with idiopathic Parkinson'sdisease, from adult patients presenting with atypical Parkinson'sdisease (Parkinson's-like disease) and from age-matched, gender-matchedhealthy control subjects. Patients with specific genetic variations mayhave copy number variations or mutations in the genes that encodeα-synuclein (PARK1), or in the gene that encodes parkin (PARKA), or inthe gene that encodes PINK 1 (PARK6), or in the gene that encodes LRRK2(PARK5). Standard protocols as disclosed herein will be used to isolatesomatic cells, for example dermal fibroblasts, from the patients, anddedifferentiate/reprogram them in to iPSC cell lines. In someembodiments a retroviral or lentiviral method is used for reprogramming.In other embodiments a method free of viral reprogramming factors isused.

Differentiation of the Parkinson's Disease-Specific iPSCs into CellularPopulations Involved in Parkinson's Disease and Parkinson's-RelatedDisease:

Following the creation, expansion, and maintenance of a bank ofParkinson's disease patient-specific iPSC cells (along with controls),the iPSCs can be differentiated to adopt a cell fate of interest,typically a cell known to be affected during the natural progression ofParkinson's disease or related diseases. For example, the iPSCs can bedifferentiated to be central or peripheral cells; neuronal or glial;autonomic or sympathetic; for example dopaminergic neurons, serotonergicneurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons,or peptidergic, neurons. Exemplary cell types include but are notlimited to neuronal and/or glial populations known to be affected inParkinson's disease and Parkinson's related diseases such as olfactorybulb neurons; glial cells such as microglia and oligodendrocytes;cholinergic neurons such as those of the nucleus basalis of Meynert;neuronal populations of the spinal cord, for example IML neurons;peripheral autonomic nervous system cells, for example superiorsympathetic cervical ganglia; neuronal populations in heart, bladder,gut, and other organs known to be affected in Parkinson's disease;cardiac cells with extrinsic and intrinsic autonomic innervation;brainstem nuclei such as pigmented nuclei; or brainstemcatecholaminergic and serotonergic nuclei.

Characterization of Differentiated Cell Lines:

In some embodiments, the iPSC line is differentiated to adopt a midbraindopaminergic cell fate. Objective measures of the dopaminergic phenotypewill be examined and include tyrosine hydroxylase positivity, and theability of the cells to synthesize and release dopamine. Additionally,neurophysiologic characteristics of dopaminergic neurons will beexamined as will the development of a neurodegenerative phenotype (invitro) reminiscent of Parkinson's disease.

Use of Parkinson's Disease-Specific Differentiated iPSC Lines forAssessing Disease Mechanisms and Screening for Therapeutic Agents Usefulfor Treatment of Parkinson's Disease and Parkinson's-Like Diseases:

In some embodiments, the cell lines and cell populations describedherein are used to study the mechanism of Parkinson's disease or relateddiseases. In some embodiments, the differentiated patient-specific cellsare compared to one or more control cells. In some embodiments, atoxicant, such as, MPTP/MPP+, 6-OHDA, rotenone, a mitochondrial toxin,or paraquat, or other such agents is applied to examine the cellularresponse of the patient-specific cell line. In some embodiments, theagent or condition produces cytotoxicity, oxidative stress, cell damage,cell degeneration, cell dysfunction, apoptosis, or mitochondrialdysfunction. Exemplary toxins include1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and structurallyrelated compounds, for example, rotenone and paraquat. Additionally, thetoxin may be a toxin or metabolite that interferes with Complex Irespiration in a cell's electron transport chain. MPTP structurallyresembles a number of known environmental agents, including well-knownherbicides such as paraquat (Di Monte, D., et al., Comparative Studieson the Mechanisms of Paraquat and 1-methyl-4-phenylpyridine (MPP+)Cytotoxicity, Biochem. Biophys. Res. Commun., 137:303-09 (1986)) andgarden insecticides/fish toxins, such as rotenone (McNaught, K. S., etal., Effects of Isoquinoline Derivatives Structurally Related to1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on MitochondrialRespiration, Biochem. Pharmacol, 51:1503-11 (1996)) that have been shownto induce dopamine cell degeneration (Brooks, A. I., et al., ParaquatElicited Neurobehavioral Syndrome Caused by Dopaminergic Neuron Loss,Brain Res., 823:1-10 (1999)). MPTP is a lipophillic molecule thatrapidly enters the brain and is taken-up into glial cells by a number ofmechanisms including monoamine (Brooks W J, et al., Astrocytes as aPrimary Locus for the Conversion MPTP into MPP+, J. Neural. Transm.76:1-12 (1989)) and glutamate (Hazell A S, et al.,1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Decreases GlutamateUptake in Cultured Astrocytes, J. Neurochem. 68:2216-19 (1997))transporters or pH-dependent antiporters (Kopin I J, Features of theDopaminergic Neurotoxin MPTP, Ann NY Acad. Sci. 648:96-04 (1992)). Oncein glial cells, MPTP is metabolized by the enzyme MAOB to the unstable1-methyl-4-phenyl-2,3-dihydropyridium (MPP+) which then rehydrogenatesor deprotonates to generate MPTP or the corresponding pyridium species,MPP+, respectively. MPP+ is then released from glia and taken up byneuronal dopamine transporters where it interferes with Complex Irespiration in the electron transport chain (Nicklas W J, et al., MPTP,MPP+ and Mitochondrial Function, Life Sci. 40:721-29 (1987)). MPP+ alsobinds to neuromelanin which is believed to contribute to itsneurotoxicity (D'Amato R J, et al., Selectivity of the ParkinsonianNeurotoxin MPTP: Toxic Metabolite MPP+Binds to Neuromelanin, Science231:987-89 (1986)). The toxicity of MPTP is determined by the responseof glial cells following drug intoxication (Smeyne M, et al.,Strain-Dependent Susceptibility to MPTP and MPP+-Induced Parkinsonism isDetermined by Glia, Glia 74:73-80 (2001)). This is supported by numerousin vitro studies (Di Monte D A, et al., Production and Disposition of1-methyl-4-phenylpyridinium in Primary Cultures of Mouse Astrocytes,Glia 5:48-55 (1992)). The cells of the present method and the toxicantare contacted, for example at 37° Celsius, for a predetermined amount oftime, after which cells in the biological sample are observed andanalyzed as discussed above.

In the present methods which use agents or conditions, the effect of theagent or condition is assessed by monitoring one or more outputparameters such as, for example, changes in ATP production, LDH release,activated caspase levels, and expression of alpha-synuclein. The resultis an analysis whereby the effect of an agent or condition on a familyof parameters permits the identification of pathways and moleculestherein affected by the disorder. The effect may be assessed bymeasuring the degree of change relative to the absence of the agent orcondition or, alternatively, relative to contacting a control cellobtained from a subject who does not have a diagnosis of a disorder ofinterest with the agent or condition. Pathways of interest include thedopamine or other neurotransmitter metabolism pathways, theO-glycosylation pathway, the caspase activation and other apoptoticpathways, signal transduction relating to Lewy bodies, mitochondrialfunction, ubiquitination, proteasome function/degradation, lysosomefunction/degradation, and/or endoplasmic reticular stress.

Human induced pluripotent stem cell (human iPSC) lines from patientswith Parkinson's disease due to a genetic basis such as a triplicationof the α-synuclein (SNCA) gene, or a homozygous mutation of the LRRK2gene, once differentiated can recapitulate key molecular aspects ofneural degeneration associated with Parkinson's disease in vitro,including α-synuclein-positive inclusions and neuritic pathology. Inother embodiment, human iPSC lines from individual presenting withidiopathic forms of Parkinson's disease can establish disease mechanismsof relevance to disease progression and useful for screening for agentsthat are disease modifying and provide tools for diagnosis of sporadicParkinson's disease.

In some embodiments the compounds listed in Table 1 can be screened foreffects in various differentiated Parkinson's disease or relateddisease-specific iPSC lines.

The cell lines can provide an entirely new experimental pre-clinicalmodel of Parkinson's disease characterized by, for example, synucleinmis-folding and/or aggregation derived from humans with geneticParkinson's disease to study cellular phenotypes and disease mechanismsunique to Parkinson's disease. The model can be also be used to screenfor new agents that slow or block progression of disease.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject methods, and are not intended to limit thescope of what is regarded as the invention.

EXAMPLES Example 1 Experimental Procedures

The following experimental procedures are utilized in the subsequentexamples.

Patient Selection:

Potential patient with familial/genetic or sporadic/idiopathic forms ofParkinson's disease (typical or atypical) and healthy volunteers wereinformed of the study through poster advertising and referrals throughpatient advocacy and disease focus organizations (Parkinson'sInstitute). Potential donors were screened for men and women between theages of 18 and 75 that had the familial or sporadic/idiopathic formsParkinson's disease. The individuals in Table 2 were identified forparticipation in the study. Participants were informed about the natureand purpose of the study and its implications and appropriatelyconsented. After informed consent was obtained, subjects underwent askin punch biopsy. This tissue was utilized for the studies for creationof cell lines.

TABLE 2 Patients identified for participation in study Cell DerivationLine Date Sex Age Phenotype/comment HUF1 Oct. 31, 2007 M 28 Healthycontrol HUF2 Nov. 28, 2007 M 62 Sporadic/Idiopathic PD HUF3 Jan. 5, 2008F 30 Healthy control HUF4 Aug. 5, 2008 M 42 Familial PD; SNCAtriplication (Iowa Kindred) HUF5 Aug. 11, 2008 F 46 Unaffected siblingto HUF4 HUF6 Sep. 16, 2008 F 60 Familial PD; LRRK2, G2019S, homozygousHUF7 Sep. 18, 2008 M 35 Offspring to HUF6 (asymptomatic); LRRK2, G2019S,heterozygous — — M 74 Familial PD; LRRK2, G2019S, heterozygous — — F 18Juvenile-onset PD; PARKIN, Ex2del, c. 102delAG — — F 40 Early-onset PD —— F 55 Sporadic/Idiopathic PD — — M 49 Sporadic/Idiopathic PD — — M 54Sporadic/Idiopathic PD — — F 49 Sporadic/Idiopathic PD — — F 54Sporadic/Idiopathic PD

Two living (and possibility the only remaining) patients withParkinson's disease secondary to documented triplication of the SNCAgene have been identified; both are members of the Iowa kindred(Singleton, A. B. et al. alpha-synuclein locus triplication causesParkinson's disease. Science 302, 841 (2003); Wszolek, Z. K. et al.Rapidly progressive autosomal dominant parkinsonism and dementia withpallido-ponto-nigral degeneration. Ann Neurol 32, 312-20 (1992)). TheHUF-4 line was created from one individual with the SNCA genetriplication.

Participants were informed about the nature and purpose of the study andits implications and appropriately consented.

Primary Cell Derivation and Culture:

After informed consent was obtained, primary Human dermal fibroblasts(HDF) from the medial arm dermus were obtained by first cleaning theregion with an alcohol swab and injecting 2-3 ml of 1% lidocaine with1:100,000 diluted epinephrine. Then, a 4 mm dermal specimen was removedwith a core punch biopsy instrument and placed in sterile PBS, while theskin defect was closed with a 4-0 nylon suture and covered with a doubleantibiotic ointment bandage. Sutures were removed after 2 weeks. Theskin tissue biopsy was washed in Ca²⁺ and Mg²⁺ free Dulbecco PBS(Invitrogen, Carlsbad, Calif.) and minced into small pieces before beingseeded onto gelatin coated 6-well cell culture flasks (Corning, Acton,Mass.) containing DMEM/F12 supplemented with 100 IU/ml penicillin, 100μg/ml streptomycin (Invitrogen), 10% FBS (DMEM/FBS culture media) andcultured at 37° C. in 5% CO₂. A minimal amount of culture media was usedto promote tissue attachment to the gelatin-coated surface (1 ml ofculture media per well). The media was brought up to 4 ml per well oncethe skin fragments attached and the media was changed every 2 days. Oncefibroblasts began to migrate out, the attached biopsy fragments and anyconnected epithelial cells were removed and the fibroblasts werecultured to 80-90% confluence (FIG. 1). This primary culture waspassaged through brief exposure to 0.15% trypsin-EDTA (Invitrogen, GrandIsland, N.Y.) and seeded into four gelatin-coated 175 cm cell cultureflasks with fresh DMEM-F12/FBS culture media. These somatic cells werecultured until they reached 90% confluence and subsequently frozen inDMEM/FBS culture media supplemented with 10% dimethyyl sulphoxide (DMSO,Sigma, St. Louis, Mo.) in aliquots of one million cells per cryovial.These somatic cells were thawed as required forreprogramming/dedifferentiation studies. As such, a bank of somaticcells were created from the donors listed in Table 2.

hESC and iPSC Maintenance:

To maintain and expand human embryonic stem cells (hESC) and inducedpluripotent stem cell (iPSC) pluripotency in vitro, colonies wereco-cultured with irradiated mouse embryonic fibroblasts (MEF) in 6-wellplates or 10 cm dishes with hESC medium (76% DMEM-F12 mediumsupplemented with 20% knock-out serum replacer (KOSR), 1% NEAA, 1%100×BME, 1% 100×L-glutamine, 1% 100× Penicillin-Streptomycin and 10ng/ml bFGF). MEFs were prepared by sacrificing pregnant CF-1 mice(Charles River Laboratories), transferring fetuses to fresh PBS andrepeating until blood was absent. The fetal heads and visceral organswere mechanically removed and the remaining carcass was transferredbetween PBS dishes until blood was removed, finishing in a dishcontaining 5 ml Trypsin. Carcass tissue was cut into small pieces usingscalpels, transferred from individual fetuses to a 15 ml centrifuge tubeand incubated in 5% CO₂ at 37° C. for 20 min. 10 ml feeder medium wasadded to neutralize the Trypsin and the solution was pipetted up anddown with a 25 ml stripper pipette. The sample was centrifuged at 1000RPM for 5 min, the supernatant was aspirated and the sample wasresuspended in 10 ml fresh feeder medium—repeated until solution wasdevoid of blood. Cells were plated at one fetus per T175 gelatin coatedflasks with 30 ml feeder medium and incubated in 5% CO₂ at 37° C. Cellswere subsequently passaged every 2-6 days (when flasks neared 90%confluency) for 5-7 passages, irradiated with 3000 rad gamma waves andthen frozen down in DMSO.

iPSC Generation using Retroviruses:

For retroviral production, 293FT cells were cultured in T175 flasks to˜90% confluence on the day of transfection. For each 293FT T175 flask,two premixes were prepared; (1) 10 ug VSVG, 15 ug Δ8.9, 10 ug retroviralvectors carrying Oct3/4, Sox2, Klf4 and c-Myc in 10 ml Opti-MEM; and (2)120 ul Lipofectamine in 5 ml Opti-MEM. The two premix solutions wereincubated for 5 min at room temperature and then mixed gently by handinversion. The resulting mix was then allowed to sit for 20 min at roomtemperature. The 293FT cells were treated with the resulting 15 ml mixand incubated in 5% CO₂ at 37° C. for 6 hours, after which thetransfection mixture was replaced with 18 ml of 10% FBS in DMEM+Glutamaxand incubated in 5% CO₂ at 37° C. for 72 hours. The supernatant washarvested in 50 ml conical tubes and spun down at 2000 rpm for 5minutes. The supernatant was filtered through a Millex-HV 0.45 filterunit and stored briefly for concentrating. To concentrate the virus, 30ml fresh viral supernatant was concentrated 100× by centrifugation at17,100 rpm for 2:20 hours at 20° C. in a Beckman Coulter Optima L-80XPUltracentrifuge and re-suspended in 300 ul of 10% FBS/DMEM. 100× viralstock was stored at −80° C.

Target fibroblasts were prepared at 1×10⁵ cells per well of a 6-wellplate. The four prepared viral supernatants were mixed to theappropriate concentrations with fresh MEF medium and supplemented with 8ng/mL polyprene and cultured with the cells overnight. The next day,cells were washed once with medium and incubated overnight with MEFmedium to allow recovery from the infection. The retroviral infectionwas repeated. After the second infection round, cells were rinsed threetimes with PBS, replaced with MEF medium and incubated for three dayswith media changes every day. On the third day, infected cells weretrypsinized and seeded at 1×10⁵ cells/10 cm dish with fresh MEF mediumand x CF1 MEFs. Eight days after infection, the medium was changed tohESC medium. Starting on day 19 post-infection, potential iPSC colonieswere identified based on morphology and manually picked and transferredto either 12- or 24-well plates with xCF1 MEF feeders in iPSC medium.

In certain experiments iPSCs were generated from fibroblasts with theuse of three factors, Oct4, Sox2, and Klf4. The generation of the HUF6line, from a patient carrying a LRRK2 homozygous mutation, the iPSCswere generated using these three 3 factors.

iPSC Generation using Lentiviruses:

For plasmid construction, the coding regions of Oct4, Sox2, Nanog,Lin28, Klf4 and cMyc were amplified by RT-PCR from the cDNA of H9 hESCs(Thomson et al., Science, 1998); the coding regions of SV40 Large T andhTERT were amplified from pBABE-puro-SV40 LT vector (plasmid 13970, T.Roberts) and pBABE-hygro-hTERT vector (plasmid 1773, R. Weinberg),respectively, purchased from Addgene. The genes were initially clonedinto pENTR™/D-TOPO® (Invitrogen) and then, together with Ubiquitin Cpromoter, recombined with p2K7-bsd lentiviral vector (Suter et al.,2006) by the Gateway (LR plus clonase enzyme mix) system (Invitrogen).Additionally, lentiviral vectors created by J. Thompson and colleagues,pSin-EF2-Oct4-Pur (Plasmid 16579, J. Thompson), pSin-EF2-Sox2-Pur(Plasmid 16577), pSin-EF2-Nanog-Pur (Plasmid 16578), andpSin-EF2-Lin28-Pur (Plasmid 16580), were purchased from Addgene.

For lentiviral production and infection, 293FT cells (Invitrogen),maintained in MEF medium supplemented with 0.5 mg/ml Geneticin(Invitrogen), were allowed to expand until reaching 90-95% confluence.One day prior to infection, fresh antibiotic-free culture medium wereadded to the cells. For each 175-cm flask, 293FT cells were transfectedwith 10 μg of plasmid DNA carrying the transgenes along with 10 μg VSVGand 15 μg Δ8.9 of the packaging plasmids. The transfection wasfacilitated by 120 ul of Lipofectamine 2000 (Invitrogen) and 15 mlopti-MEM (Invitrogen) for 6 hours and then replaced with 17 ml of freshMEF medium without antibiotics. After 3 days, the viral supernatant wascollected by spinning and passing through a Millex-HV 0.45 um filterunit (Millipore). The viral supernatants were concentrated byultracentrifugation (Optima L-80XP, Beckman Coulter) at 17,100 RPM for2.2 hours at 20° C.

For lentiviral infection and iPSC generation, one day beforetransduction, human fibroblast cells were seeded at 8×10⁴ cells per wellof a 6-well plate. Next day, equal volumes (0.5 ml) of freshsupernatants, supplemented with 8 ng/ml polyprene, carrying each of thesix lentiviruses were mixed and added to the growing fibroblasts. 24hours later, the viral supernatants were washed with PBS and replacedwith fresh MEF medium. 5 days post-transduction, the cells wereresuspended with trypsin, counted, and seeded, at 5×10⁴ cells/dish, onto10-cm dishes pre-plated with irradiated CF1 feeders. After overnightincubation, the MEF medium was replaced with hESC medium, andthereafter, the medium was changed every other day or every day, ifrequired. hESC-like colonies started to appear among hundreds ofbackgrounds colonies around 14-20 days post-transductions. The colonieswere manually picked and transferred to 24-well plates pre-plated withCF1 feeders. Colonies that continued to expand and maintained theirhESC-like morphology were further passaged into larger vessels; whereas,those that failed to expand and/or exhibited early signs ofdifferentiation were discarded. FIG. 2 illustrates the creation of theHUF1 line (see Table 2) using a lentiviral-mediated protocol andestablishment of colonies.

iPSC Generation Using Non-Virally Mediated Direct Delivery ofReprogramming Proteins:

Plasmids are constructed by amplifying human ESC cDNA for Oct4, Sox2,Klf4, and c-Myc and cloned into pcDNA3.1/myc-HisA (Invitrogen)constructs. The protein extracts are prepared by transfecting HEK 293cells using a reagent such as Lipofectamine. About 2 ug of plasmid DNAis transfected per about 4×10̂5 cells and stably expressing cell linesare established by selection with an antibiotic such as with about 500ug/ml neomycin (G418). To prepare protein extracts, cells are washed inPBS, centrifuged at about 400×g, and lysed in ˜1 volume of cold lysisbuffer (about 100 mM HEPES, about 50 mM NaCl, about 5 mM MgCl2, about 2mM dithiothreitol, and protease inhibitors) for about 45 min on ice.Cells are sonicated on ice and lysates are sedimented at about 15,000 gfor about 15 min at about 4° C. to pellet debris. Supernatant is thenfiltered through a ˜0.2 um membrane, aliquoted, and snap-frozen oncrushed dry ice.

For protein transduction, human fibroblasts are incubated with all fourprotein factors for about 8 hrs a week up to about 6 weeks and culturedin DMEM media supplemented with about 2 mM L-glutamine, about 1 mMbeta-mercaptoethanol, ˜1× non-essential amino acids, about 20% fetalbovine serum, about 1500 U/ml LIF. After ˜6 weeks, cells are dissociatedand transferred to inactivated mouse feeder cells and cultured with hESCmedia consistent of KO-DMEM, about 20% KSOR, about 2 mM L-glutamine,about 1 mM beta-mercaptoethanol, ˜1× non-essential amino acids, about 16ng/ml bFGF until iPSC colonies are formed.

Reverse Transcription, Pre-Amplification and RT-PCR:

RNA was purified using QIAGEN Quick Prep-Mini kit or cell sortingdirectly into the pre-amplification reaction mix. Samples were thenreverse transcribed and pre-amplified with 5 ul CellsDirect 2× ReactionMix, 10 ul Superscript III TR/Platinum Taq Mix (Invitrogen, CellsDirectOne-Step qRT-PCR kit), 2.5 ul of 0.5× pooled primers and probes, 1.5 ulTE Buffer (QIAGEN) and 0.1 ul SUPERaseIn (Applied Biosystems). RT-PCRwas performed in 20 ul volumes with 10 ul ABI 2× Reaction Mix (AppliedBiosystems), 1 ul FAM probe, 1 ul VIC probe, 0.5-2 ul of pre-amplifiedsample, were mixed with water and amplified as follows: Pre-ampthermocycle: 95° C. for 10 min, 18 cycles of 95° C. for 15 seconds, 60°C. for 4 min, and then hold at 4° C.

Immunocytochemistry and Alkaline Phosphatase Staining:

Alkaline Phosphatase (AP) staining was performed with Vector® RedAlkaline Phosphatase Substrate Kit I (Vector Laboratories, CA),according to the manufacturer's protocol. For immunocytochemistry, cellswere fixed in 4% paraformaldehyde/PBS for 10 minutes, washed twice withPBS, and blocked with 1-3% donkey, goat or chicken serum in PBS for 1hour—all procedures were done at room temperature. For nuclear orintracellular staining, after fixation, the cells were permeabilizedwith 0.3% Triton-X100 for 30 minutes at room temperature. Subsequently,the primary antibodies were added to PBS and incubated for 1 hour atroom temperature or overnight at 4° C. The cells were then washed withPBS before fluorescent-conjugated secondary antibodies were added andincubated for an hour at room temperature. Finally, the cells wererinsed with PBS three times and counter stained with DAPI. Stainedsamples were imaged directly on a LEICA inverted microscope or, if thesamples were on coverslips, were mounted in PVA-DAVCO overnight and thenimaged on an inverted confocal microscope. Primary antibodies and theirdilutions were used as follows: Oct4 (diluted at 1:100, Santa Cruz),Sox2 (1:200, Millipore), SSEA1 (1:200, Millipore), SSEA4 (1:200,Millipore), TRA1-60 (1:200, Millipore), TRA1-81 (1:200, Millipore),Nanog (1:100, Abcam), α-Fetoprotein (1:200, Abcam), 131II Tubulin(1:200, Abcam), α-Smooth muscle actin (1:200, Abcam), Vasa (1:200,Abcam) Nestin (1:200, Santa Cruz), Doublecortin (1:200, Santa Cruz),Tyrosine hydroxylase (1:500, Pel-Freez Biologicals). Secondaryantibodies were raised in either donkey, goat or rabbit with conjugates:Alexa 488-conjugated anti-rabbit IgG (1:500, Invitrogen), Alexa594-conjugated anti-rabbit (1:500, Invitrogen), Alexa 647-conjugatedanti-rabbit (1:500, Invitrogen), Alexa 488-conjugated anti-mouse IgM(1:500, Invitrogen) and Alexa 488 anti-mouse IgG (1:500, Invitrogen),FITC anti-Mouse conjugated, Cy3 anti-Rabbit, and Cy5 anti-Goat.

Directed Midbrain Dopaminergic Differentiation:

To direct differentiation of pluripotent cell colonies towards amidbrain dopaminergic cell fate, two or three hESC or iPSC colonies weremechanically harvested and gently dissected into 4-6 pieces per colony.Cells were then plated onto a 6 cm co-culture dish with irradiated MS-5(xMS-5) stromal cells (MS-5 cells were expanded in MS-5 stromal cellculture medium: 455 ml alpha-MEM, 2 mM L-glutamine, 50 ml heatinactivated FBS, Pen-strep) and cultured for 16 days in SerumReplacement Media (15% KOSR in KO-DMEM), with media changes every twodays. After 16 days, media was changed to N2+/+ (Progesterone 20 nM,Putrescine 100 uM, Sodium Selenite 30 nM, Insulin 5 ug/ml, Transferrin0.1 mg/ml in DMEM-F12). At day 28, neural rosettes in the colonies werepassaged through mechanical micro-dissection and transferred ontoPoly/Laminin coated (15 ug/ml polyornithine, 1 ug/ml laminin) 6-wellplates and cultured for one week in 200 ng/ml Sonic Hedgehog (SHH), 100ng/ml Fibroblast Growth Factor 8 (FGF-8), 20 ng/ml Brain DerivedNeurotrophic Factor (BDNF), and 0.2 mM Ascorbic Acid (AA) in N2+/+media. On day 35, when cultures are approximately 80% confluent,colonies were passaged through digestion in Ca²⁺/Mg²⁺ free HBSS at roomtemperature for 1 hour and subsequent mechanical dissociation. Removedcell aggregates were centrifuged at 200 g for 5 minutes, resuspended andplated on Poly/Laminin coated dishes at 50-100×10³ cells/cm² in N2+/+media with the previously noted, day 28, growth factors. On day 42,differentiation was induced for 8 days through growth factor withdrawalby changing media to N2+/+ with 20 ng/ml BDNF, 20 ng/ml Glial DerivedNeurotrophic Factor (GDNF), 1 ng/ml Transforming Growth Factor P3(TGF-133), 1 mM Dibutyryl Cyclic Adenosine Monophosphate (cAMP) and 0.2mM AA in N2+/+ media. On day 50, cell cultures were harvested foranalysis.

Generation of ES-derived dopaminergic neurons is demonstrated in FIG. 3.Dopaminergic neuron production from the hESC H9 line is demonstrated bythe accumulation of TH-positive, NeuN positive neurons. These neuronsform fasciculated bundles of neurites that course between neuronal cellaggregates. This protocol and the like can be used to differentiate thenewly generated hiPSC lines.

Phenotypes of the differentiated cells can be evaluated usingimmunofluorescent staining for markers associated with midbrain-specificdopaminergic neurons as well as other neuronal and glial cell subtypes.For example, multiple staining for generic neuronal and glial markersmay provide information on the overall makeup of differentiated cellpopulations. Neuronal markers that may be used include Map2 (immatureand mature neurons), type III beta tubulin (immature neuron),doublecortin (immature), and NeuN (mature). Glial markers that may beused include glial fibrillary acidic protein (astrocytes), S100-beta(astrocytes), NG2 (oligodendrocytes), and GalC (oligodendrocytes). Thefraction of neurons exhibiting a midbrain-specific dopaminergicphenotype may be further evaluated by staining for tyrosine hydroxylase(TH) and evaluating cells for co-expression of midbrain-consistentmarkers such as TH, aromatic amino acid decarboxylase, Grk2, and absenceof markers for non-midbrain neuronal subtypes such glutamatetransporter, GAD or dopamine beta hydroxylase.

Human iPSCs from patients with genetic variations are compared tocontrols. The cells are tested for markers of (1) general cytotoxicityin cell viability assays, (2) apoptosis, (3) oxidative stress, and (4)mitochondrial function. These markers are to determine if there aredifferences in the survival and viability between cells SNCAtriplication and controls. The cells are also tested for severalparameters such as ATP, LDH release, and activated caspase levels usingluminescent assays on a multi-well microplate luminometer. Furthermore,the expression of α-synuclein is assessed by qRT-PCR andimmunohistochemistry to investigate α-synuclein pathology in thiscellular model.

Alternatively, different feeder cells are used and/or the recombinantgrowth factors are modified to produce a neuronal dopaminergic phenotype(Yan, Y. et al. Directed differentiation of dopaminergic neuronalsubtypes from human embryonic stem cells. Stem Cells 23, 781-90 (2005);Park, C. H. & Lee, S. H. Efficient generation of dopamine neurons fromhuman embryonic stem cells. Methods Mol Biol 407, 311-22 (2007)). Inanother alternative, the differentiated cells are exposed to varioustoxicants known to induce neurodegeneration and cellular damage such asMPTP and paraquat.

Bisulfite Sequencing:

To determine methylation status, indicative of active transcription,bisulfite sequencing was performed on genomic DNA isolated from hESCsand iPSCs, grown on feeder-free media, with Methyl Easy Xceed Rapid DNABisulfite Modification Kit (Human Genetic Signatures, Sydney, New SouthWales, Australia) per manufacturers directions. The promoter regions ofOct3/4 and Nanog were amplified by PCR, as described by Deb-Rinker atal. (Deb-Rinker et al., 2005). The PCR products were subcloned intopCR2.1 TOPO (Invitrogen), and twelve clones from each sample wereanalyzed by sequencing with M13 universal primer.

In Vitro Differentiation:

To investigate in vitro differentiation, cell culture medium wasreplaced with ˜80% KO DMEM, 20% fetal bovine serum (FBS), 1%100×L-glutamine, 1% 100× betamercaptoethanol (BME), 1% 100×non-essential amino acids (NEAA), and 1% 100× Penicillin-Streptomycin.After culturing for the desired time period, cells were either examinedby immunocytochemistry or harvested for protein or RNA analysis.

In Vivo Teratoma Formation and Immunohistochemistry:

To determine iPSC potential to form all three germ layers in vivo, hESCsand iPSCs cells were harvested from 6-well or 10 cm plates through briefCollagenase IV treatment and transferred to 200 ul of hESC medium. Thecells were either grafted subcutaneously behind the neck or in the hindlimp muscles of female SCID mice (Charles River). After 8-10 weekspost-transplantation, grafts were dissected and fixed with 4%paraformaldehyde/PBS overnight. The tissues were then paraffin embedded,sectioned and stained with Masson's Trichrome, Mayer's Mucicarmine,Saffron O and Hematoxylin and Eosin.

Karyotyping:

To prepare the karyotyping metaphase spread, 10 μl/mL colcemid was addedto the cell culture and incubate for up to 2 hours. The growth mediumwas removed and collected while the cells were rinsed with HBSS. Cellswere treated with 2 mL trypsin and re-incubated at 37° C. for 5-7 min.The collected colcemid medium from the earlier step was re-applied tothe cells to neutralize the trypsin and resuspend the cells. Theresulting solution was centrifuged at 1000 RPM for 6 min and thesupernatant was partially aspirated and resuspended in the nativesolution by flicking the tube. 5 drops of pre-warmed hypotonic solutionwere slowly added against the side, while flicking with a finger, until1 ml had been added. Volume was then brought to 2 mL with hypotonicsolution. The sample was incubated at 37° C. for 7 min and thencentrifuged at 1000 RMP for 6 min. Medium was added to resuspend thecells. To fix the cells, 5 drops of fixative were slowly added againstthe side of the tube and the volume was brought to 2 mL with fixative.Cells were “reverse bubbled” to fully mix the cells and then the cellswere left to fix for 30 min at room temperature. After fixing, thesample was centrifuged, aspirated, and resuspended with the finger asbefore. Any clumps were removed by vacuum from the side of tube, and 2ml of fixative were added to the tube. Sample was then “reversebubbled”, let stand for 20 min at room temperature, and thencentrifuged, aspirated, and resuspended with finger, as before. Samplewas resuspended in 2 ml of fixative and then transferred ontopre-cleaned slides in ˜100 ul drops, left to dry overnight, and thenanalyzed on a SKY microscope.

Characterization of Phenotype:

To characterize the differentiated cell lines, several assays will beused. Differentiated iPSCs will be examined for signs of spontaneouspathology starting with measures of dopaminergic cell abundance andsurvival (% dopaminergic neurons after differentiation) and generalmorphological attributes such as inclusion bodies, or dystrophicneurites. Markers which implicate selected mechanisms of pathology willalso be evaluated including 1) apoptosis (TUNEL, caspase activation), 2)necrosis (CytoTox-Glo), 3) oxidative stress (glutathione, ROS and4-HNE), 4) mitochondrial dysfunction (MitoExpress, ATP content); 5)Protein aggregation of α-synuclein will be assessed with antibodiesagainst total, phosphorylated and nigrated α-synuclein to detectaggregates, inclusion bodies, and neuritic pathology. Cells fromaffected individuals will be compared to those from healthy controls.

Example 2 Derivation of Control Human Fibroblasts (HUF1 Line)

The HUF1 line was created from a healthy control volunteer. Similar tothe techniques presented in Example 1, after informed consent wasobtained, the patient's skin was cleaned with an alcohol swab, and a fewmilliliters of 1% lidocaine with epinephrine diluted 1:100,000 wasinjected into the skin to achieve local anesthesia. A 4 mm core punchbiopsy instrument was used to remove a piece of skin and the specimenwas placed on saline Patient participation lasted approximately 1 hour.The skin defect was closed with 4-0 nylon suture and a double antibioticointment covered bandaid was applied to the wound. Sutures were removedin 2 weeks. A primary somatic fibroblast cell culture was establishedfrom the skin biopsy. The skin tissue biopsy was washed in Ca and Mgfree Dulbecco PBS (Invitrogen, Carlsbad, Calif.) and minced into smallpieces. Tissue pieces were seeded onto gelatin coated 6-well cellculture flasks (Corning, Acton, Mass.) containing DMEM supplemented with100 IU/ml penicillin, 100 μg/ml streptomycin (Invitrogen), 10% FBS(DMEM/FBS culture media) and cultured at 37 C in 5% CO2. A minimalamount of culture media was used to promote tissue attachment to thegelatin coated surface (1 ml of culture media per well). The media wasbrought up to 4 ml per well once the skin fragments attached and themedia was changed every 2 days. Once fibroblasts began to grow, theattached biopsy fragments were removed and the fibroblasts were becultured to confluence. This primary culture was passaged through briefexposure to 0.25% trypsin-EDTA (Invitrogen, Grand Island, N.Y.) andseeded into several new T175 cell culture flasks with fresh DMEM/FBSculture media. These somatic cells were cultured until they reached ˜90%confluence and then frozen down at passage 1 in DMEM/FBS culture mediasupplemented with 10% dimethyyl sulphoxide (DMSO, Sigma, St. Louis, Mo.)in aliquots of one million cells per cryovial. These somatic cells werethawed as required for further reprogramming research. Manuallydissected human skin biopsy fragments demonstrated attachment to agelatin coated surface and primary human fibroblasts outgrowths formedover a 2 week period (see FIG. 3).

Lentiviral Transduction of Primary Human Fibroblasts FUGW UbC-GFPLentiviral Supernatant Production:

A 90% confluent T175 of 293T cells (p13) were Lipofectamine 2000transfected with 10 ug VSV-G, 15 ug Δ8.9 and 10 ug FUGW. Plasmids werepremixed in 10 ml of Opti-MEM and 120 ul of Lipofectamine was premixedin 5 ml Opti-MEM. Following a 5 minute incubation at room temperaturethe plasmids and lipofectamine were mixed together and incubated afurther 20 minutes at room temperature to allow DNA-lipofectaminecomplexes to form. Following this incubation the plasmid-lipofectaminemixture was gently laid over the 293T cells and incubated for 6 hours ina 37 C incubator. Following this transfection the mixture was replacedwith 15 mls of DMEM+10% FBS and incubated for 3 days in the lentiviralroom. Following the 3 day incubation the viral supernatant was spun downat 2000 rpm for 5 min and filtered through a 0.4 um Millex-HV filterunit and then used fresh.

Infection of Primary Human Fibroblasts (HUF1):

Primary human fibroblasts (p4) were transduced at 40-50% confluence on a6 well plate. This level of confluence allowed for the determination ofthe post-transductional viability based on the ability of the cells toproliferate. 2 ml of four-fold dilute fresh supernatant wasappropriately diluted in DMEM+10% FBS, mixed with polybrene to a finalconcentration of Bug/ml and incubated with the HUF1 cells overnight at37 C. Following overnight transduction the cells were washed twice inPBS and incubated for 3 days in DMEM+10% FBS in regular 37 C incubator.At the end of the 3 day incubation the cells were examined for GFPexpression both by fluorescence microscopy and FACS analysis. Resultsare shown in FIG. 4.

Transduction of Primary Human Fibroblasts with Reprogramming Factors:

The cDNAs for the open reading frames (ORFs) of human LIN28, cMYC andKLF4 genes were obtained by direct PCR of human ES cell cDNA and aftersequence verification, the cDNAs for each gene were cloned into a 2K7lentiviral vector backbone (Ubiquitin C promoter), which was derivedfrom a recombinant HIV-1-based, replication-defective vector,pLENTI-Block-iT-DEST (Invitrogen). The OCT4, SOX2 and NANOG lentiviralvectors were provided courtesy of Professor Jamie Thomson (pSIN vectorbackbone, EF1a promoter). These combination of the OCT4, SOX2, NANOG andLIN28 genes represent the Thomson reprogramming factors (Yu, Vodyanik etal. 2007). We also investigated if the addition of cMYC and KLF4 couldincrease the iPS reprogramming efficiency. The 293T cell line(Invitrogen) was used to produce transgene-expressing lentivirussupernatant which was used fresh. Lentiviral transductions of the humanprimary fibroblasts (HUF1, p2) were carried out with cells in attachment(˜80% confluence/2 ml/well of 12-well gelatin-coated plate) using aequal mixture of the viral supernatants (2 ml total volume) in thepresence of polybrene (8 μg/ml final concentration, Sigma). One HUF1sample was transduced with just the Thomson reprogramming factors (OCT4,SOX2, NANOG and LIN28) and a second sample was transduced with theThomson reprogramming factors plus cMYC and KLF4 (six factors total).Following overnight incubation with lentivirus, the human somatic cellswere cultured for 72 hours and then trypsinized and transferred togelatin coated T175 flasks. Cells were incubated in MEF conditionedembryonic stem (ES) cell media until the formation of iPS colonies withhuman embryonic stem cell morphology. MEF conditioned media was replacedevery two days. For ES media DMEM/F12 culture media was supplementedwith 0.1 mM non-essential amino acids, 1 mM L-glutamine, 0.1 mMB-mercaptoethanol, 8 ng/ml basic fibroblast growth factor (FGF) and 20%knock-out serum replacer (KSR).

Results:

Colonies formed after 4 weeks; those with characteristics that resembledhESCs were manually dissected into small pieces and cultured on freshMEFs, 8 from the Thomson factor transduced HUF1 cells and 8 from theThomson factor+cMyc and Klf4 transduced HUF1 cells. Some coloniesproduced outgrowths and formed lines with characteristic morphology asshown in FIGS. 5-7.

Example 3 Derivation, Characterization and Differentiation of HumaniPSCs from a Parkinson's Disease Patient with a Triplication of the SNCAGene

iPSC lines were generated from human fibroblasts of a patient (obtainedby skin biopsy) with Parkinson's disease due to a SNCA triplication(HUF4 line) and his unaffected female sibling (HUF5 line), using aretroviral system with factors known to reprogram somatic cells,according to the methods of Example 1.

FIG. 8 illustrates SNCA triplication as confirmed in HUF4 iPSC clone 17line, by qPCR. Quantitative real-time PCR (qRT-PCR) analysis for theSNCA gene was performed using 40 ng of genomic DNA and SYBR® Green PCRMaster Mix. Reactions were performed in triplicate, wherein each targetregion was co-amplified with an internal control (GAPDH) using the ABI7000 RT-PCR system (Applied Biosystems). Primers used were:

SNCA Exon 3 forward primer TGACAAATGTTGGAGGAGCASNCA Exon 3 reverse primer CTGGGCTACTGCTGTCACAC GAPDH forward primerTGGGCTACACTGAGCACCAG GAPDH reverse primer GGGTGTCGCTGTTGAAGTCA

Gene copy number was determined with the standard curve method. Theratio of target gene compared to an internal control gene of 0.8-1.0 isequivalent to a normal copy number of 2 (alleles). A ratio of 1.8-2.2 isconsidered a copy number of 4 and is equivalent to a triplication of thetarget gene. FIG. 8 depicts the gene dosage SNCA Exon3

FIG. 9 illustrates the untransduced fibroblasts used for the generationof the HUF4 and HUF5 lines. The HUF4 and HUF5 lines were stained andimaged to assess pluripotency. Pluripotency stains show successfulreprogramming/dedifferentiation of the HUF4 clone 17 (FIG. 10) and HUF5clone 2 (FIG. 11) lines.

To further assess pluripotency status of the cell lines, in vitrodifferentiation of the HUF4 clone 17 and HUF5 clone 2 lines were carriedout. FIG. 12 illustrates that both HUF4 and HUF5 cells formed embyroidbodies, functional cardiac myocyte differentiation, and the three germlayers, as assessed by staining for germ layer-specific markers.Endoderm formation was assessed by staining for α-fetalprotein; mesodermformation was assessed by staining for α-SMA and desmin; and ectodermformation was assessed by staining for β-III-tubulin, doublecortin (DCX)and tyrosine hydroxylase (TH) (FIG. 12).

Karyotype analyses of the HUF4 clone 17 and HUF5 clone 2 lines werecarried out to assess for any aberrant, macroscopic chromosomalabnormalities. The results of the analysis indicated no translocations,triplications, or deletions (FIG. 13).

The methylation status of the Nanog and Oct4 promoters was determined bycarrying out bisulfite sequencing, as described in the methods ofExample 1. These two endogenous transcription factors are related topluripotency. FIG. 14 shows the methylation status of the two lines atdifferent stages.

Relative telomerase activity was assessed in untransduced HUF4 and HUF5cell lines, pluripotent iPSC lines, and pluripotent hESC lines, anddifferentiated iPSC lines HUF4 and HUF5. The results are presented inFIG. 15.

Teratoma sectioning was carried out, as an in vivo demonstration of theformation of the three germ layers, endoderm, mesoderm, and ectoderm.The results are presented in FIG. 16 for the HUF4 clone 17 line.

FIG. 17-21 illustrate qPCR expression of endogenous and exogenoustranscription factors following reprogramming in HUF4 and HUF 5 lines(parental, iPSCs, differentiated) as well as control H9 and Hela cells.Endogenous and exogenous expression of Oct4, Sox2, cMyc, and Klf4expression are examined. Primers used for the qPCR were as follows:

Primers for Exogenous Transcription Factor Expression Analysis:

pMXs-AS3200: ttatcgtcgaccactgtgctgctg(Used as Reverse primer for all exogenous transcription factor analysis expression) Oct4-Forward:ccccagggccccattttggtacc Sox2-Forward: ggcacccctggcatggctcttggctcKlf4-Forward: acgatcgtggccccggaaaaggacc cMyc-Forward:caacaaccgaaaatgcaccagccccag

Primers for Endogenous Transcription Factor Expression Analysis:

Oct4-Forward: ccccagggccccattttggtacc Oct4-Reverse:cctagctcctcccctccccctgtc Sox2-Forward: ggcacccctggcatggctcttggctcSox2-Reverse: cctcttttgcacccctcccatttccc Klf4-Forward:acgatcgtggccccggaaaaggacc Klf4-Reverse: tgattgtagtgctttctggctgggctcccMyc-Foward: ttgaggggcatcgtcgcgggaggctg cMyc-Reverse:cgagaggaccccgtggatgcagag

Known techniques were used to differentiate the generated cell lines(from Example 2) into dopaminergic neuronal cells (FIG. 3, Methods inExample 1). Neural differentiation was carried out and neural inductionparameters were examined. Further midbrain specification was thenpromoted by addition of specific factors.

Markers of neural induction were examined at day 28 and day 50. TH,Nestin markers were visualized and analyzed. FIG. 22 shows the resultsand indicates midbrain dopaminergic neuron presence.

FIGS. 23 and 24 show images of the time course of neural induction.Clover-like rosette patterns are visible in the HUF4 clone 17 line (FIG.23) and HUF5 clone 2 line (FIG. 24).

Expression of known neural fate markers were examined in the H9 ESCs,and the differentiated HUF4 and HUF5 lines, and are depicted in FIG. 25.

FIG. 26 illustrates α-synuclein staining of HUF4 cells.

A 96-gene panel of qPCR was run to compare the quantitative geneexpression in H9 ESCs, HUF4 and HUF5 cell lines. The primer sets listedin Table 3 were purchased from Invitrogen and examined on parentalfibroblast, iPSC, ESC, and dopaminergic differentiated cell lines. Theexperiment was performed to detect quantitative differences in classesof genes involved in ES cell function, neural progenitor function,dopamine neuron function, netrin receptor function, slit receptorfunction, ephrin receptor function, and pathological function.

TABLE 3 Taqman primers purchased from Invitrogen for analysis on 96-genearray Oct4 Lmx1b GAD2 DNAJA1 Klf4 Msx1 GAD1 HSPB1 SOX2 Msx2 SPON1 robo4DNMT3B Ngn2 CALB1 robo3 c-Myc Engrailed 1 BDNF robo2 LIN28 Engrailed 2SNCG robo1 TERT ALDH1A1 Nkx6.1 MAOB NANOG NR4A2 Chat MAOA GAPDH B3GAT1(beta III MNX1 HMOX2 tubulin) (Hb9) CTNNB1 Pitx3 TPH2 HMOX1 EEF1A1 THPNMT CASP9 CENTB3 DDC DBH HSPA1A (CENTRIN) Sox2 (TYPE2) SLC6A3 UCHL1GSTP1 Gata6 Pax2 PARK7 MT2A Sox17 Pax6 PARK2 MT1A Pdx-1 FoxA1 SNCA GPX1Brachyury FoxA2 (Hnf-3b) Olig2 SOD2 Gata-1 SHH GFAP NOX1 NCAM NTN1 DCCISL-1 PRPH gdnf NTN1 FOXD3 (Peripherin 1) Nes RET HTRA2 PROX1 Sox1SLC18A2 ATP13A2 NKX2.1 Otx2 DRD2 LRRK2 NEFL (Neurofilament LightProtein) Lmx1a KCNJ6 PINK1 Map2

Example 4 Derivation, Characterization and Differentiation of HumaniPSCs from a Parkinson's Disease Patient Carrying a Homozygous Mutationat the LRRK2 Locus

iPSC lines were generated from human fibroblasts of a female patient(obtained by skin biopsy) with Parkinson's disease due to homozygousmutation at the LRRK2 locus (HUF6 line, Table 2) and her asymptomaticson (HUF7 line, Table 2), using a retroviral system with factors knownto reprogram somatic cells, according to the methods of Example 1.

FIG. 27 shows the glycine to serine G2019S mutation in the HUF6 parentalfibroblasts and the reprogrammed/de-differentiated iPSC line HUF6 (clone3), but not the HUF4 line. The HUF6 iPSC line was derived from the HUF6parental fibroblasts and with the use of three factors: Oct4, Sox2, andKLF4. Staining for pluripotency markers are presented in FIG. 28.

Karyotype analysis was carried out with the HUF6 iPSC line and ispresented in FIG. 29. No gross chromosomal abnormalities were observed.

Growth on matrigel and feeders was examined and is presented in FIG. 30.The figure shows growth and expansion of HUF6 iPS cells on matrigel andfeeders, indicative of stem cell-like morphology.

A 20-day protocol was carried out to differentiate the HUF6 line todopaminergic neurons. The 20-day protocol is presented below. Cells werevisualized along the time course of differentiation and are presented inFIG. 31. Between Day 1 and Day 5 of the differentiation protocol, Nogginwas expressed and the small molecule SB431542 was applied in SRM media.Between Day 5 and Day 9 of the differentiation protocol Sonic hedgehogwas expressed in N2 media. Between Day 9 and Day 12, BDNF, AA, SHH, andFGF8b were expressed in N2 media. Between Day 12 and Day 20, BDNF, AA,GDNF, TGF-13, and cAMP were expressed in N2 media. (protocol in Chamberset al. 2009). FIG. 32 depicts the differentiation of HUF6-iPSCs intodopaminergic neurons after 20 days.

Example 5 Non-Viral Derivation of Human iPSCs from a Parkinson's DiseasePatient Carrying a Homozygous Mutation at the LRRK2 Locus

iPSC lines are generated from human fibroblasts of a female patient(obtained by skin biopsy) with Parkinson's disease due to homozygousmutation at the LRRK2 locus using a non-viral system with factors knownto reprogram somatic cells, according to the methods of Example 1. Thisnon-viral system is free of viral reprogramming/dedifferentiatingfactors. Fibroblasts are reprogrammed and differentiated intodopaminergic neurons. Making the iPSCs free of viral reprogrammingfactors involves use of cre-recombinase excisable viruses. The cells arethen differentiated into brainstem nuclei, olfactory neurons,dopaminergic neurons and cholinergic neurons and the lines aremaintained. If any of the differentiated cell lines display spontaneousformation of α-synuclein mediated aggregation, the lines are used as atool for screening candidate agents (such as those in Table 1) for theirability to dis-aggregate or prevent aggregation of α-synuclein. Suchcells lines can be used as a tool for screening candidate agents (suchas those in Table 1) for their ability to dis-aggregate or preventaggregation of α-synuclein. As shown in FIG. 33, in vitro and ex vivomethods for rapid screening of anti-aggregation compounds are used forthe screening. FIG. 33 shows: (A) Inhibition of fibrillation ofα-synuclein upon incubation with a specific inhibitor, detected byThioflavin T fluorescein and (B-C) confirmed by electron microscopy ofα-synuclein fibrils. (D-E) Diminished Thioflavin S deposits (which labelaggregate protein) detected in paraquat-treated mouse brain withCatechol (250 uM), indicative of treatment-induced dissolution ofaggregate structure.

While embodiments of the present methods have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the invention. It should be understood thatvarious alternatives to the embodiments of the invention describedherein may be employed in practicing the claimed methods. It is intendedthat the following claims define the scope of the invention and thatmethods and structures within the scope of these claims and theirequivalents be covered thereby.

What is claimed is: 1.-65. (canceled)
 66. A method of producing adifferentiated cell line, the method comprising: a. producing inducedpluripotent stem cells (iPSCs) from somatic cells harvested from asubject diagnosed with a disease, wherein the somatic cells carry agenetic variation that is associated with the disease; and b.differentiating the iPSCs towards a cell type that is associated withthe disease, wherein the differentiated iPSCs display a phenotype whichis characteristics of the cell type, thereby resulting in thedifferentiated cell line.
 67. The method of claim 66 wherein the somaticcells are fibroblasts.
 68. The method of claim 66, wherein the geneticvariation is a copy number variation.
 69. The method of claim 68,wherein the genetic variation is a triplication.
 70. The method of claim66, wherein the genetic variation is a deletion, an insertion, a complexmulti-state variant, a substitution, a transition, a transversion, or aduplication, of one or more nucleotides in a gene that is associatedwith the disease.
 71. The method of claim 66, wherein the disease is aneurodegenerative disease.
 72. The method of claim 66, wherein thedisease is Parkinson's disease, Alzheimer's disease, dementia, an autismspectrum disorder, susceptibility to viral infection, diffuse Lewy bodydisease or any other Lewy body disorder or synucleinopathy, corticobasaldegeneration, encephalitis lethargica, multiple system atrophy,pantothenate kinase-associated neurodegeneration (Hallervorden-Spatzsyndrome), progressive supranuclear palsy, vascular Parkinsonism, Wilsondisease, hereditary pancreatitis, glomerulonephritis, human systemiclupus erythematosus, paraneoplastic syndrome, frontotemporal dementiawith Parkinsonism chromosome 17, Huntington's disease, spinocerebellarataxias, amytropic lateral sclerosis, or Creutzfeld Jakob disease. 73.The method of claim 66, wherein the disease is Parkinson's disease orParkinson's-like disease.
 74. The method of claim 73, wherein thegenetic variation is in a gene selected from PARK1 (SNCA orα-synuclein), PARK2 (Parkin), PARK5 (UCHL1), PARK6 (PINK1), PARK7(DJ-1), PARK8 (LRRK2), and PARK 11 (GIGFY2).
 75. The method of claim 74,wherein the gene is PARK1 (SNCA or α-synuclein).
 76. The method of claim74, wherein the gene is PARK8 (LRRK2).
 77. The method of claim 75wherein the variation is triplication of the SNCA gene.
 78. The methodof claim 76, wherein the genetic variation comprises a G2019S mutation.79. The method of claim 68, wherein the disease is 12q14 microdeletionsyndrome, 15q13.3 microdeletion syndrome, 15q24 recurrent microdeletionsyndrome, 16p11.2-p12.2 microdeletion syndrome, 17q21.3 microdeletionsyndrome, 1p36 microdeletion syndrome, 1q21.1 recurrent microdeletion,1q21.1 recurrent microduplication, 1q21.1 susceptibility locus forThrombocytopenia-Absent Radius (TAR) syndrome, 22q11 deletion syndrome(Velocardiofacial/DiGeorge syndrome), 22q11 duplication syndrome,22q11.2 distal deletion syndrome, 22q13 deletion syndrome(Phelan-Mcdermid syndrome), 2p15-16.1 microdeletion syndrome, 2q33.1deletion syndrome, 2q37 monosomy, 3q29 microdeletion syndrome, 3q29microduplication syndrome, 6p deletion syndrome, 7q11.23 duplicationsyndrome, 8p23.1 deletion syndrome, 9q subtelomeric deletion syndrome,adult-onset autosomal dominant leukodystrophy (ADLD), Angelman syndrome(Type 1), Angelman syndrome (Type 2), ATR-16 syndrome, AZFa, AZFb,AZFb+AZFc, AZFc, Cat-Eye Syndrome (Type I), Charcot-Marie-Tooth syndrometype 1A (CMT1A), Cri du Chat Syndrome (5p deletion), early-onsetAlzheimer disease with cerebral amyloid angiopathy, familial adenomatouspolyposis, hereditary liability to pressure palsies (HNPP), Leri-Weilldyschondrostosis (LWD)-SHOX deletion, Miller-Dieker syndrome (MDS),NF1-microdeletion syndrome, Pelizaeus-Merzbacher disease, Potocki-Lupskisyndrome (17p11.2 duplication syndrome), Potocki-Shaffer syndrome,Prader-Willi syndrome (Type 1), Prader-Willi syndrome (Type 2), RCAD(renal cysts and diabetes), Rubinstein-Taybi syndrome, Smith-Magenissyndrome, Sotos syndrome, split hand/foot malformation 1 (SHFM1),steroid sulphatase deficiency (STS), WAGR 11p13 deletion syndrome,Williams-Beuren syndrome (WBS), Wolf-Hirschhorn syndrome, or Xq28(MECP2) duplication.
 80. The method of claim 66, wherein the iPSCs aredifferentiated into neural cells.
 81. The method of claim 66, whereinthe iPSCs are differentiated towards a midbrain dopaminergic cell type.82. The method of claim 66, wherein the iPSCs are differentiated toadopt the cell type within 20 days.
 83. The method of claim 66, whereinthe iPSCs are produced without the use of a retrovirus or a lentivirus.84. The method of claim 66, wherein the iPSCs are produced with a methodcomprising the use of three factors.
 85. The method of claim 84, whereinthe three factors are OCT4, SOX2, and KLF4.
 86. The method of claim 66,wherein the phenotypic characteristics are in cell viability, cellularchemistry, cellular function, mitochondrial function, cell aggregation,cell morphology, cellular protein aggregation, gene expression, cellularsecretion, or cellular uptake.
 87. The method of claim 66, wherein thephenotypic characteristics changes when the cell line is in contact witha putative therapeutic agent.
 88. The method of claim 66, wherein thecell type is a cell type that is affected in the natural progression ofthe disease.
 89. A method of screening a putative therapeutic agentusing the cell line produced by the method of claim
 66. 90. A method oftreating a subject with the disease using the putative therapeutic agentscreened by the method of claim 89.